System, device and method for high-throughput multi-plexed detection

ABSTRACT

The present invention relates to a system, device, and method for the high throughput multiplexed detection of a wide number of compounds. The invention comprises of a microwell array coupled to a capture agent array to form a plurality of interfaces between a microwell and a set of immobilized capture agents. The set of capture agents comprises a plurality of distinguishable features, with each feature corresponding to the detection of a particular compound of interest. In certain embodiments, each microwell is configured to contain a single cell. The invention is therefore capable of performing a high throughput analysis of single cell profiles, including profiles of secreted compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/294,853, filed Mar. 6, 2019, which is a continuation of U.S.application Ser. No. 15/290,874, filed Oct. 11, 2016, which is acontinuation of U.S. application Ser. No. 14/629,164, filed Feb. 23,2015, which is a continuation of U.S. application Ser. No. 14/562,061,filed Dec. 5, 2014, which is a continuation of international applicationnumber PCT/US2013/056454, filed Aug. 23, 2013, which claims the benefitunder 35 U.S.C. § 119(e) of U.S. provisional application No. 61/692,895,filed Aug. 24, 2012, and of U.S. provisional application No. 61/779,299,filed Mar. 13, 2013, each of which is herein incorporated by referencein its entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under NIH UO1 CA164252,NIH 4R00 CA136759, NIH U54CA143868, NIH RO1 GM084201, and NIH U54CA143798, awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Secreted proteins including cytokines, chemokines, and growth factorsrepresent important functional regulators mediating a range of cellularbehavior and cell-cell paracrine/autocrine signaling, e.g., in theimmunological system (Rothenberg, 2007, Nat. Immunol 8(5):441-4), tumormicroenvironment (Hanahan and Weinberg, 2011, Cell 144(5):646-74), orstem cell niche (Gnecchi et al., 2008, Circ. Res 103(1):1204-19).Detection of these proteins is of great value not only in basic cellbiology but also for disease diagnosis and therapeutic monitoring.However, because of coproduction of multiple effector proteins from asingle cell, referred to as polyfunctionality, it is biologicallyinformative to measure a panel of secreted proteins, or secretomicsignature, at the level of single cells. Recent evidence furtherindicates that a genetically identical cell population can give rise todiverse phenotypic differences (Niepel et al., 2009, Curr Opin Chem Biol13(5-6):556-561). Nongenetic heterogeneity is also emerging as apotential barrier to accurate monitoring of cellular immunity andeffective pharmacological therapies (Gascoigne and Taylor, 2008, CancerCell 14(2):111-22; Cohen et al., 2008, Science 322(5907):1511-6),suggesting the need for practical tools for single cell analysis ofproteomic signatures.

Fluorescence-activated cell sorting (FACS) represents thestate-of-the-art for single cell analysis (Sachs et al., 2005, Science308(5721):523-9). FACS is typically used to detect and sort cellphenotypes by their surface markers. It has been extended to thedetection of intracellular proteins (Sachs et al., 2005, Science308(5721):523-9; Kotecha et al., 2008, Cancer Cell 14(4):335-43; Irishet al., 2004, Cell 118(2):217-28), including cytokines within thecytoplasm, by blocking vesicle transport (Prussin, 1997, Clin Immunol17(3):195-204). However, intracellular cytokine staining (ICS) is not atrue secretion analysis, and it also requires cell fixing, which meansthe cells are no longer alive after flow cytometric analysis and cannotbe recovered for further studies. A further disadvantage to ICS is thespectral overlap and the possibility of non-specific binding ofintracellular staining antibodies, which will ultimately preventaccurate multiplexing over the current capability of 12-plexing. Themainstay of real single cell secretion analysis to date is a simpleapproach called ELISpot, a plate based cell culture assay using standardELISA detection, which detects the secretion footprint of individualcells using an immunosandwich-based assay (Sachdeva and Asthana, 2007,Front Biosci 12:4682-95). Immune cells are loaded into a microtiterplate that has been precoated with a layer of primary antibody. Afterincubation, secreted proteins are captured by the antibodies locatedproximal to the cells, giving rise to spots indicative of a single cellsecretion footprint (Stratov et al., 2004, Curr Drug Targets5(1):71-88). Recently, a variant of ELISpot, called FLUOROSpot, whichexploits two fluorescent dyes to visualize protein secretion footprints,enabled a simultaneous dual function analysis, though this technique islimited to low multiplexing capabilities. Highly multiplexedmeasurements of proteins secreted from a population of cells can be doneusing an encoded bead assay such as the Illumina VeraCode system(Henshall and Gorfain, 2007, Genet Eng Biotechnol News 27(17): 1) orantibody microarrays manufactured using a pin-spotting technique (Chenet al., 2007, Nat Methods 4(5):437-44; Liotta et al., 2003, Cancer Cell3(4):317-25). However, these highly multiplexed technologies cannotperform single cell measurements. Microfabricated chips have emerged asa new category of single cell analytic technologies (Wang and Bodovitz,2010, Trends Biotechnol 28(6):281-90; Cheong et al., 2009, Sci Signal2(75):p12; Love et al., 2006, Nat Biotechnol 24(6):703-7; Lee et al.,2012, Integr Biol (Camb) 4(4):381-90; Rowat et al., 2009, Proc Natl AcadSci USA 106(43):18149-54; Lecault et al., 2011, Nat. Methods8(7):581-6). A prototype microchip has demonstrated the feasibility ofthe multiplexed protein secretion assay and revealed significantpolyfunctional heterogeneity in phenotypically similar immune cells frompatients (Shin et al., 2011, Biophys J 100(10):2378-86; Ma et al., 2011,Nat Med 17(6):738-43), pointing to the urgent need for single cellsecretion profiling in clinical diagnosis and therapeutic monitoring.However, these microchips either lack sufficient throughput ormultiplicity or require sophisticated operation, precluding widespreadapplication in cell biology and clinical evaluation of cellularfunctions. These technologies cannot perform highly multiplexed proteinanalysis on single cells. For example, thus far there is no technologyavailable to perform high-content (>1000 cells) and highly multiplexed(>35 proteins) measurement of secreted proteins at the single celllevel.

Thus, there is a need in the art for a device and method for multiplexanalysis of a wide number of compounds from single cells. The presentinvention satisfies this unmet need.

SUMMARY OF THE INVENTION

The present invention includes a device for the multiplexed detection ofa plurality of compounds from single cells comprising a microwell arrayand a capture agent array. The microwell array comprises a plurality ofindividual microwells in uniform arrangement, at least some of theplurality of individual microwells having a length of greater than 50 μmand configured to contain an isolated single cell in a sub-nanolitervolume of contents. The capture agent array comprises a plurality ofimmobilized capture agents, each immobilized capture agent capable ofspecifically binding to one of the plurality of compounds. Theimmobilized capture agents are arranged in uniform capture agent sets,where each capture agent set comprises a plurality of isolated featuresat spatially identifiable locations, each isolated feature comprising atleast one immobilized capture agent. The microwell array and captureagent array are coupled to form a plurality of enclosed interfaces, eachenclosed interface comprising a microwell and a capture agent set suchthat the contents of each microwell are accessible to all of theisolated features of at least one set, thereby accessible to all of theimmobilized capture agents.

In one embodiment, each of the plurality of isolated features has adistinguishable spatial localization. In one embodiment, each of theplurality of isolated features is a feature selected from the groupconsisting of a line, shape, and dot. For example, in one embodiment,the shape of each isolated feature is distinguishable from the shape ofall other isolated features.

In one embodiment, at least some of the plurality of microwells is ahigh aspect ratio rectangular well, having dimensions of about 1-2 mm inlength and about 5-50 μm in depth.

In one embodiment, the plurality of compounds comprise at least onecompound selected from the group consisting of a protein, peptide,peptide fragment, cell surface receptor, nucleic acid, hormone, antigen,and growth factor. In one embodiment the plurality of compounds compriseat least one protein secreted from a single cell contained within amicrowell.

In one embodiment, the plurality of capture agents comprise at least onecompound selected from the group consisting of an antibody, protein,peptide, peptide fragment, and nucleic acid.

In one embodiment, at least one isolated feature comprises one or moreimmobilized capture agents, wherein each immobilized capture agentwithin the isolated feature has an associated secondary capture agentwith a different detectable label.

In one embodiment, each microwell is rectangular with a length of about10-2000 μm, a width of about 10-100 μm, and a depth of about 10-100 μm.

In one embodiment, each capture agent set comprises about 10-100isolated features, each isolated feature comprising at least oneimmobilized capture agent that specifically binds to one compound.

In one embodiment, each isolated feature has a width of about 25-30 μm.In one embodiment, each isolated feature is separated from anotherisolated feature of the same set at a distance of about 25 μm for apitch size of about 50 μm or more.

In one embodiment, the capture agent array comprises greater than 10different capture agents, thereby allowing for the detection of greaterthan 10 different compounds. In one embodiment the capture agent arraycomprises greater than 40 different capture agents, thereby allowing forthe detection of greater than 40 different compounds.

In one embodiment, the microwell array comprises a microwell density ofabout 200 microwells per cm² to about 20,000 microwells per cm².

The present invention also includes a method of spatially encodedmultiplexed detection of a plurality of compounds from a single cell,the method comprising providing a microwell array comprising a pluralityof individual microwells in uniform arrangement; applying a fluid to asurface of the microwell array such that a sub-nanoliter volume of thefluid comprising a single cell flows into at least one microwell;providing a capture agent array comprising a plurality of immobilizedcapture agents, each capture agent capable of specifically binding toone of the plurality of compounds, where the immobilized capture agentsare arranged in capture agent sets, wherein each capture agent setcomprises a plurality of isolated features at spatially identifiablelocations, each isolated feature comprising at least one immobilizedcapture agent; and contacting the microwell array with the capture agentarray to form a plurality of enclosed interfaces, each enclosedinterface comprising a microwell and a capture agent set such that thefluid within each microwell is accessible to all of the isolatedfeatures of a set and is thereby accessible to all of the of immobilizedcapture agents. The method further comprises providing suitableconditions to allow for the binding of the plurality of compounds to theimmobilized capture agents to form immobilized capture agent-compoundcomplexes; removing the capture agent array from the microwell array;contacting the capture agent array with a plurality of labeled secondarycapture agents, wherein each labeled secondary capture agentspecifically binds to a formed immobilized capture agent-compoundcomplex, to form immobilized capture agent-compound-labeled secondarycapture agent complexes; detecting the presence of the detectable labelon the capture agent array; and correlating the presence of thedetectable label on the capture agent array with the presence of atleast one compound.

In one embodiment each of the plurality of isolated features is afeature selected from the group consisting of a line, shape, and dot.For example, in one embodiment, the shape of each isolated feature isdistinguishable from the shape of all other isolated features.

In one embodiment, the fluid applied to the microwell surface comprisesa cell. In one embodiment, the fluid flows into an individual microwellby gravitational force alone.

In one embodiment, the plurality of compounds comprise at least onecompound selected from the group consisting of a protein, peptide,peptide fragment, cell surface receptor, nucleic acid, hormone, antigen,and growth factor. In one embodiment, the plurality of compoundscomprise at least one protein secreted from a single cell containedwithin a microwell.

In one embodiment, the plurality of capture agents comprise at least onecompound selected from the group consisting of an antibody, protein,peptide, peptide fragment, and nucleic acid.

In one embodiment, at least one isolated feature comprises more than oneimmobilized capture agent, wherein each immobilized capture agent withinthe isolated feature has an associated secondary capture agent with adifferent detectable label.

In one embodiment, each microwell is rectangular with a length of about10-2000 μm, a width of about 10-100 μm, and a depth of about 10-100 μm.

In one embodiment, each capture agent set comprises about 10-100isolated features, each isolated feature comprising at least oneimmobilized capture agent that specifically binds to one compound.

In one embodiment, each isolated feature has a width about 25-30 μm. Inone embodiment, each isolated feature is separated from another isolatedfeature of the same set at a distance of about 25 μm.

In one embodiment, the capture agent array comprises greater than 10different capture agents, thereby allowing for the detection of greaterthan 10 different compounds. In one embodiment, the capture agent arraycomprises greater than 40 different capture agents, thereby allowing forthe detection of greater than 40 different compounds.

In one embodiment, the microwell array comprises a microwell density ofabout 200 microwells per cm² to about 20,000 microwells per cm².

In one embodiment, applying the fluid to the microwell array surfaceproduces a plurality of individual microwells which comprise a singlecell.

In one embodiment, the spatial location of the detected detectable labelon the capture agent array is correlated to the identity of at least oneof the plurality of compounds and to the individual microwell from whichthe compound was detected. In one embodiment, the shape of the detecteddetectable label on the capture agent array is correlated to theidentity of at least one of the plurality of compounds. In oneembodiment, the spectral properties of the detected detectable label onthe capture agent array is correlated to the identity of at least one ofthe plurality of the compounds. In one embodiment, the spatial locationof the detected detectable label and spectral properties of the detecteddetectable label on the capture agent array is correlated to theidentity of one of the plurality of compounds and to the individualmicrowell from which the compound was detected. In one embodiment, thespatial location of the detected detectable label, shape of the detecteddetectable label, and the spectral properties of the detected detectablelabel on the capture agent array is correlated to the identity of one ofthe plurality of compounds and to the individual microwell from whichthe compound was detected.

In one embodiment, the method assays the phenotype of a plurality ofsingle cells within the sample by detecting 5 or more compounds secretedby the single cells.

In one embodiment, applying the suspension to the microwell arraysurface produces a plurality of individual microwells which eachcomprise a single cell, thereby providing a high throughput method ofmultiplexed detection of compounds secreted by a plurality of singlecells.

In one embodiment, the combination of compounds detected in anindividual microwell is indicative of the phenotype of the single cellcontained within the microwell. In one embodiment, the phenotype of thecell defines the cell as a cancer cell. In one embodiment, the phenotypeof the cell defines the cell as a metastasizing cancer cell. In oneembodiment, the phenotype of the cell defines the aggressiveness of acancer cell.

In one embodiment, the plurality of single cells comprises a populationof immune cells and the method assays the heterogeneity of the immunecells. In one embodiment, the phenotype of one or more single cellsindicates the progression of a disease or delineates individual diseasestages.

The present invention also includes a method of spatially and spectrallyencoded multiplexed detection of a plurality of compounds from a singlecell, the method comprising providing a microwell array comprising aplurality of individual microwells in uniform arrangement; applying afluid to a surface of the microwell array such that a sub-nanolitervolume of the fluid comprising a single cell flows into at least onemicrowell; providing a capture agent array comprising a plurality ofimmobilized capture agents, each capture agent capable of specificallybinding to one of the plurality of compounds, where the immobilizedcapture agents are arranged in capture agent sets, wherein each captureagent set comprises a plurality of isolated features at spatiallyidentifiable locations, each isolated feature comprising more than oneimmobilized capture agent; and contacting the microwell array with thecapture agent array to form a plurality of enclosed interfaces, eachenclosed interface comprising a microwell and a capture agent set suchthat the fluid within each microwell is accessible to all of theisolated features of a set and is thereby accessible to all of the ofimmobilized capture agents.

The method further comprises providing suitable conditions to allow forthe binding of the plurality of compounds to the immobilized captureagents to form immobilized capture agent—compound complexes; removingthe capture agent array from the microwell array; contacting the captureagent array with a plurality of labeled secondary capture agents,wherein each secondary capture agent is labeled with one of a pluralityof detectable labels, where each secondary capture agent is configuredto bind to a immobilized capture agent-compound complex at an isolatedfeature to form an immobilized capture agent-compound-secondary agentcomplex, such that the immobilized capture agent-compound-secondarycapture agent complexes of an isolated feature each have a spectrallydistinct label; detecting the presence of the plurality of detectablelabels on the capture agent array; and correlating the spatial locationand spectral properties of each detected detectable label on the captureagent array with the presence of at least one compound.

In one embodiment each of the plurality of isolated features is afeature selected from the group consisting of a line, shape, and dot.For example, in one embodiment, the shape of each isolated feature isdistinguishable from the shape of all other isolated features.

In one embodiment, the fluid applied to the microwell surface comprisesa cell. In one embodiment, the fluid flows into an individual microwellby gravitational force alone.

In one embodiment, the plurality of compounds comprise at least onecompound selected from the group consisting of a protein, peptide,peptide fragment, cell surface receptor, nucleic acid, hormone, antigen,and growth factor. In one embodiment, the plurality of compoundscomprise at least one protein secreted from a single cell containedwithin a microwell.

In one embodiment, the plurality of capture agents comprise at least onecompound selected from the group consisting of an antibody, protein,peptide, peptide fragment, and nucleic acid.

In one embodiment, at least one isolated feature comprises more than oneimmobilized capture agent, wherein each immobilized capture agent withinthe isolated feature has an associated secondary capture agent with adifferent detectable label.

In one embodiment, each microwell is rectangular with a length of about10-2000 μm, a width of about 10-100 μm, and a depth of about 10-100 μm.

In one embodiment, each capture agent set comprises about 10-100isolated features, each isolated feature comprising at least oneimmobilized capture agent that specifically binds to one compound.

In one embodiment, each isolated feature has a width about 25-30 μm. Inone embodiment, each isolated feature is separated from another isolatedfeature of the same set at a distance of about 25 μm.

In one embodiment, the capture agent array comprises greater than 10different capture agents, thereby allowing for the detection of greaterthan 10 different compounds. In one embodiment, the capture agent arraycomprises greater than 40 different capture agents, thereby allowing forthe detection of greater than 40 different compounds.

In one embodiment, the microwell array comprises a microwell density ofabout 200 microwells per cm² to about 20,000 microwells per cm².

In one embodiment, applying the fluid to the microwell array surfaceproduces a plurality of individual microwells which comprise a singlecell.

In one embodiment, the spatial location of the detected detectable labeland spectral properties of the detected detectable label on the captureagent array is correlated to the identity of one of the plurality ofcompounds and to the individual microwell from which the compound wasdetected. In one embodiment, the spatial location of the detecteddetectable label, shape of the detected detectable label, and thespectral properties of the detected detectable label on the captureagent array is correlated to the identity of one of the plurality ofcompounds and to the individual microwell from which the compound wasdetected.

In one embodiment, the method assays the phenotype of a plurality ofsingle cells within the sample by detecting 5 or more compounds secretedby the single cells.

In one embodiment, applying the suspension to the microwell arraysurface produces a plurality of individual microwells which eachcomprise a single cell, thereby providing a high throughput method ofmultiplexed detection of compounds secreted by a plurality of singlecells.

In one embodiment, the combination of compounds detected in anindividual microwell is indicative of the phenotype of the single cellcontained within the microwell. In one embodiment, the phenotype of thecell defines the cell as a cancer cell. In one embodiment, the phenotypeof the cell defines the cell as a metastasizing cancer cell. In oneembodiment, the phenotype of the cell defines the aggressiveness of acancer cell.

In one embodiment, the plurality of single cells comprises a populationof immune cells and the method assays the heterogeneity of the immunecells. In one embodiment, the phenotype of one or more single cellsindicates the progression of a disease or delineates individual diseasestages.

The present invention also includes a system for the multiplexeddetection of a plurality of compounds from single cells comprising adevice comprising a microwell array and a capture agent array, and aplurality of secondary capture agents. The microwell array comprises aplurality of individual microwells in uniform arrangement, at least someof the plurality of individual microwells configured to contain a singlecell in a sub-nanoliter volume of contents. The capture agent arraycomprises a plurality of immobilized capture agents, each immobilizedcapture agent capable of specifically binding to one of the plurality ofcompounds, where the immobilized capture agents are arranged in uniformcapture agent sets, wherein each capture agent set comprises a pluralityof isolated features at spatially identifiable locations, each isolatedfeature comprising at least one immobilized capture agent. The microwellarray and capture agent array are coupled to form a plurality ofenclosed interfaces, each enclosed interface comprising a microwell anda capture agent set such that the contents of each microwell areaccessible to all of the isolated features of at least one set, therebyaccessible to all of the immobilized capture agents. Each secondarycapture agents comprises a detectable label and is configured to bind toa immobilized capture agent-compound complex formed at an isolatedfeature by the binding of a compound of the plurality of compounds to animmobilized capture agent of the plurality of immobilized captureagents.

In one embodiment, each of the plurality of isolated features has adistinguishable spatial localization. In one embodiment, each of theplurality of isolated features is a feature selected from the groupconsisting of a line, shape, and dot. For example, in one embodiment,each isolated feature is distinguishable from the shape of all otherisolated features.

In one embodiment, at least some of the plurality of microwells is ahigh aspect ratio rectangular well, having dimensions of about 1-2 mm inlength and about 5-50 μm in depth.

In one embodiment, the plurality of compounds comprise at least onecompound selected from the group consisting of a protein, peptide,peptide fragment, cell surface receptor, nucleic acid, hormone, antigen,and growth factor. In one embodiment the plurality of compounds compriseat least one protein secreted from a single cell contained within amicrowell.

In one embodiment, the plurality of capture agents comprise at least onecompound selected from the group consisting of an antibody, protein,peptide, peptide fragment, and nucleic acid.

In one embodiment, at least one isolated feature comprises one or moreimmobilized capture agents, wherein each immobilized capture agentwithin the isolated feature has an associated secondary capture agentwith a different detectable label.

In one embodiment, each microwell is rectangular with a length of about10-2000 μm, a width of about 10-100 μm, and a depth of about 10-100 μm.

In one embodiment, each capture agent set comprises about 10-100isolated features, each isolated feature comprising at least oneimmobilized capture agent that specifically binds to one compound.

In one embodiment, each isolated feature has a width of about 25-30 μm.In one embodiment, each isolated feature is separated from anotherisolated feature of the same set at a distance of about 25 μm for apitch size of about 50 μm or more.

In on embodiment, the capture agent array comprises greater than 10different capture agents, thereby allowing for the detection of greaterthan 10 different compounds. In one embodiment the capture agent arraycomprises greater than 40 different capture agents, thereby allowing forthe detection of greater than 40 different compounds.

In one embodiment, the microwell array comprises a microwell density ofabout 200 microwells per cm² to about 20,000 microwells per cm².

In one embodiment, the detectable label is selected from the groupconsisting of a fluorescent label, radioactive label, ferromagneticlabel, paramagnetic label, luminescent label, electrochemiluminescentlabel, phosphorescent label, and chromatic label.

In one embodiment, each of the plurality of secondary capture agentscomprise the same detectable label.

In one embodiment, each secondary capture agent is labeled with one of aplurality of detectable labels, where each secondary capture agent isconfigured to bind to a immobilized capture agent-compound complex at anisolated feature to form an immobilized capture agent-compound-secondaryagent complex, such that the immobilized captureagent-compound-secondary capture agent complexes of an isolated featureeach have a spectrally distinct label.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIGS. 1A-1C show a set of images depicting the structure of an exemplaryhigh-throughput multiplexed single cell secretomic assay. FIG. 1Adepicts a schematic illustration showing integration of a high-densitycapture agent array chip and a subnanoliter microchamber array chip forhigh-throughput multiplexed protein secretion assay at the single celllevel. FIG. 1B is a scanned fluorescence image showing high uniformityof protein loading across the entire capture agent microarray (1 in.×2in.). Fluorescently labeled bovine serum albumin (FITC-BSA) was used inthis test. FIG. 1C is a photograph stitched from a large number ofindividual pictures collected by an automated, motorized phase contrastmicroscope. Together it covers the entire subnanoliter microchamber chipthat was loaded with human immune cells (U973). Scale bar 2 mm. Thefirst enlarged image shows a column of microchamber array (scale bar 300μm). The second enlarged image shows individual cells loaded inmicrochambers (scale bar 50 μm).

FIGS. 2A-2B illustrate an exemplary protein panel assayed in anexemplary device of the invention. FIG. 2A is a list of all 22 proteinsassayed in single cell microchips and their functions in humanphysiology. FIG. 2B is a set of titration curves obtained usingrecombinant proteins. A total of 18 antibody pairs were validated and 4others were left out in the titration curves due to the lack of workingrecombinants. Fluorescence intensity represents the original photoncounts averaged from 16 spots for each protein. Error bars indicate3×SD.

FIGS. 3A-3D show a set of images depicting the results of single-cellsecretomic analysis on U87 cell lines. FIG. 3A is an image depicting arepresentative region of the scanned image showing the raw data ofsingle cell secretomic measurement. Three subpanels on the right areoptical micrograph, fluorescence image, and overlay for 16microchambers. FIG. 3B depicts a heat map that shows the profile of 14proteins secreted from 1278 single cells (U87). Each row is a singlecell and each column corresponds to a protein of interest. FIG. 3C is aset of scatter plots showing fluorescence intensity measured for sixselected proteins (FGF, VEGF, MIF, IL-6, IL-8, MCP-1) versus the numberof cells in a microchamber. (*P<0.05, **P<0.01, ***P<0.001) FIG. 3Ddepicts the population kinetics for U87 cell line. Control (MEM medium),secretion supernatant from population at different time points (0 h, 1h, 2 h, 3 h, 6 h, 9 h, 12 h, 24 h).

FIGS. 4A-4D show a set of images depicting the results of experimentsdemonstrating the correlation between protein secretion profiles andcellular migration for A549 cells. FIG. 4A is an image depictingrepresentative optical images showing three single cells (n=384) before(0 h) and after (24 h) protein secretion assay. FIG. 4B is a scatterplot showing the fluorescence intensity corresponding to IL-8 secretionversus migration distance of individual cells (P<0.05). FIG. 4C is ascatter plot showing a similar analysis on MCP-1 (P=0.14). FIG. 4D is ascatter plot showing a similar analysis on IL-6 (P=0.75). Each dotrepresents a single cell.

FIGS. 5A-5E show a set of images depicting the single-cell secretomicanalysis of primary tumor cells from patients. FIG. 5A depicts aschematic for the procedure for processing tissue specimens, preparationof single cell suspension and application of primary cells to thesubnanoliter microchamber array chip. FIG. 5B depicts a representativeregion of the scanned image for patient 1. FIG. 5C and FIG. 5D depictheat maps showing single-cell secretomic signatures of primary tumorcells from two patients (patients 1 and 2), respectively. The data arepresented as a result of unsupervised hierarchical clustering analysis.FIG. 5E is a set of scatter plot matrices showing protein-proteincorrelation in single cells. Each subpanel is the scatter plot showingthe level of a protein versus the other in all single cells measured.The proteins are indicated at the diagonal line. The correlationcoefficient is computed as R via a linear regression analysis. Theentire matrix is color-coded by red (positive correlation) and blue(negative correlation). The color intensity is proportional to the Rvalue.

FIG. 6 is a set of images depicting the assembly of an exemplarycomplete single cell secretomic analysis device. A high-density antibodyarray glass slide and a 5440-microchamber PDMS slab were clampedtogether with two transparent plates using a device housing systemcontaining a clamp with exerted spring force.

FIG. 7 is a graph depicting the evaluation of the whole chip uniformityof the capture agent array. Quantification of fluorescence intensityacross the flow patterned poly-L-lysine slide (3 cm×2 cm) revealexcellent uniformity of the immobilized proteins (FITC-BSA), whichensures the validity of using this high-density array technology toassess single cell heterogeneity.

FIG. 8 is a graph depicting the distribution of the number of cellsacross a whole microchip. Four experiments were performed with differentquantities of cell suspensions (cell density: 10⁶ cells/mL).

FIGS. 9A-9C show a set of images depicting the single-cell secretomicanalysis on U937 cell lines. FIG. 9A is an image depicting arepresentative region of the scanned image showing the raw data ofsingle cell secretomic measurement. Three subpanels on the right areoptical micrograph, fluorescence image and overlay for 14 microchambers.FIG. 9B is a heat map that shows the profile of 14 proteins secretedfrom 551 single cells (U937). Each row is a single cell and each columncorresponds to a protein of interest. FIG. 9C is a pair of scatter plotsshowing fluorescence intensity measured for four selected proteins (IL8,MCP-1, RANTES and TNFa) versus the number of cells in a microchamber.The cells were stimulated with 20 μg/mL of PMA to differentiate tomacrophage and then challenged by 1 mg/mL of LPS to become activatedright before they were loaded onto the microchip to secretion analysis.

FIG. 10 is a set of heat maps that show the profile of 14 proteinssecreted from chambers with U87 cell line. Each row is a cell chamberand each column corresponds to a protein of interest. Zero cells(n=1821), single cells (n=1278), two cells (n=544), three cells (n=214),four cells (n=100), and five cells (n=35).

FIG. 11 is a graph depicting the population kinetics for U87 cell line.Control (MEM medium), secretion supernatant from population at differenttime points (0 hr, 1 hr, 2 hr, 3 hr, 6 hr, 9 hr, 12 hr, 24 hr)

FIG. 12 is an image depicting the average. Average signal of U87 cellswithin the single cell platform approaching bulk patterns (top) and the24 hour bulk secretion profile of U87 cells (bottom)

FIG. 13 is a set of images depicting the results of a control experimentmeasuring U937 population secretion. Proteins secreted from a largepopulation of U937 cells were measured by a conventional antibodymicroarray (upper panels). Quantification of all 23 proteins are shownin the lower panel.

FIG. 14 is a heat map showing protein secretion profiles of single A549cells at their basal level without stimulations.

FIG. 15 is a set of images depicting the results of a control experimentmeasuring A549 population secretion. Proteins secreted from a largepopulation of A549 cells were measured by a conventional antibodymicroarray (upper panels). Quantification results of all 23 proteins areshown in the lower panel.

FIG. 16 is a heat map showing the correlation between A549 single cellmigration distance and its corresponding protein secretion signals.

FIG. 17 is a set of histogram plots of individual proteins measured onthe sample from Patient 1. (light gray=zero cells, dark gray=singlecells)

FIG. 18 is a set of scatter plots showing fluorescence intensitymeasured for eight selected proteins from Patient 2. (FGF, IL-6, IL-8,MCP-1, MIF, PDGF, RANTES, TNF-α)

FIG. 19 is a heat map depicting single cell protein secretion profilingon the sample from Patient #3, a transitional meningioma patient.

FIG. 20 is a set of scatter plots and protein correlation analysis ofthe single cell secretion data obtained from Patient 3.

FIG. 21 is a set of images depicting an exemplary device of theinvention.

FIG. 22 is a schematic depicting an exemplary microwell array andcapture probe array of the invention.

FIGS. 23A-23B show a set of schematics depicting exemplary isolatedfeatures of the invention. FIG. 23A depicts an exemplary set of isolatedfeatures. Features are measured at 25 microns per line and have a 25micron spacing to realize a pitch size of 50 microns per non-parallelline set. FIG. 23B is a schematic depicting exemplary isolated featuresof the invention, with features having distinct geometries.

FIG. 24 is a set of images depicting an ultra-high-density antibodymicroarray. The shape of each microwell can be varied (i.e., square vs.diamond shapes) and this array can be interfaced with single-cellmicrowell cell capture chip to conduct single-cell high-plex proteinprofiling. This array is fabricated by cross-flow patterning techniqueand can also be fabricated by micro-scale printing techniques such asmicrospotting and inject printing.

FIG. 25 is a set of images depicting detection of cells withinindividual microwells (bottom right) and detection of the presence ofparticular compounds of interest (bottom center).

FIG. 26 is a correlation map analysis of a 45-plexed cytokine,chemokine, and extracellular protein (e.g, growth factor) 3-colorspectral detection using the device of the invention.

FIG. 27 is a diagram depicting the use of three different primaryantibodies, and three different detection antibodies, each labeled witha different fluorescent label, to provide a spatial and spectral (i.e.fluorescent colorimetric) encoding of capture of single cell compoundssingle-cell assay of the invention.

FIG. 28 is a set of images depicting the imaging of three differentdetectable labels on the same view of the antibody array, demonstratingthe spectral encoding of the array and the ability for the multiplexeddetection of a large number of compounds.

FIG. 29 is a set of images depicting the uniformity in imaging of thedevice using a multitude of different detectable labels. Scannedfluorescence images (mixed and separate) showing the result of multipleantibody co-immobilization on poly-l-lysine glass slide. The insetfigure shows high uniformity of protein coating across the entirecapture agent slide (C.V. <5% in 1 in.×2 in area). Fluorescently labeledbovine serum albumin (488-BSA, 532-BSA, 647-BSA respectively) were usedin this test.

FIG. 30 is a table of an exemplary panel of 42 different cytokines,extracellular proteins, growth factors, and antigens, using 14 spatiallocations (lines) and 3 colors per location.

FIG. 31 depicts the results of an experiment demonstrating thespecificity of the assay via the shown non-crossreactivity of thecapture agents, where a sample containing only EGF results in thepresence of fluorescence in only the location/wavelength combinationcorresponding to EGF.

FIGS. 32A-32C show a set of calibration curves obtained with recombinantproteins for groups for the compounds in the 488 group (FIG. 32A), the532 group (FIG. 32B) and the 635 group (FIG. 32C). These curves can beused to quantify the concentration of each cytokine in a sample, basedon a detected intensity.

FIG. 33 depicts the raw data from the 488 nm imaged wavelength using thespectral assay of the invention.

FIG. 34 depicts the raw data from the 532 nm imaged wavelength using thespectral assay of the invention.

FIG. 35 depicts the raw data from the 635 nm imaged wavelength using thespectral assay of the invention.

FIG. 36 depicts the combined raw data from the 488 nm, 532 nm, and 635nm imaged wavelengths using the spectral assay of the invention.

FIG. 37 depicts raw data of cytokine detection using the populationbased micro-ELISA.

FIG. 38 is a graph depicting the comparison of population-basedmicro-ELISA and the single-cell assay of the invention.

FIG. 39 depicts the results of the experiments of using the 45-plexedsingle cell assay comparing single-cell secretions of non-stimulatedversus LPS (100 ng/mL) stimulated cells. Data is presented as ahistogram (top), heat maps (middle) and 2-d bar graph of the averagedsignals (bottom).

FIG. 40 is a table comparing the single-cell assay of the invention(SCMA) with intracellular cytokine staining (ICS) in detection ofcytokines in nonstimulated (control) and LPS stimulated cells.

FIG. 41 is a graph comparing the single-cell assay of the invention(SCMA) with intracellular cytokine staining (ICS) in detection ofcytokines in nonstimulated (control) and LPS stimulated cells.

FIG. 42 is a set of graphs depicting the polyfunctionality of cells byillustrating the number of secreted cytokines per cell, as measured bythe single-cell assay of the invention, for control and stimulatedcells.

FIG. 43 depicts a workflow illustration of the 45-plexed single cellprotein secretion profiling system.

FIGS. 44A-44D depict the results of experiments demonstrating U937macrophage single cell results on the 45-plexed protein secretionprofiling platform. FIG. 44A depicts a comparison of the U937 derivedmacrophage single cell protein secretion results with population cellsecretion results. FIG. 44B depicts a comparison of protein secretionfrequency obtained from single cell secretion platform and ICS(intracellular cytokine staining). FIG. 44C is a set of graphsdemonstrating similar cell subpopulation definitions defined by IL-8 andMCP-1 protein secretion results both by SCMA (left) and ICS (right).FIG. 44D depicts U937 macrophage single cell polyfunctionality analysisbased on their protein secretion results.

FIGS. 45A-45C depict the results of experiments demonstrating themacrophage response upon TLR 4 ligand LPS stimulation. FIG. 45A depictsheatmaps showing comparison between untreated and LPS stimulated U937monocyte derived macrophage protein secretion profiles. FIG. 45B depictsvisualization of ingle cell secretion results with VISNE. FIG. 45Cdepicts visualization of individual proteins (MIF, IL-8, MCP-1, RANTES,MIP-1a, MIP-1b) secretion results with VISNE.

FIGS. 46A-46D depict the results of experiments demonstrating themacrophage response upon different TLR ligands (LPS, PAM3, poly IC)stimulation. FIG. 46A depicts heatmaps showing protein secretionprofiles comparison between untreated and stimulated (LPS, PAM3, polyIC) U937 monocyte derived macrophage. FIG. 46B depicts a heat mapshowing the frequency of U937 macrophage cell that secrete anyindividual protein under different stimulations. FIG. 46C depicts thevisualization of single cell results with VISNE. FIG. 46D depicts thevisualization of individual proteins (MIF, IL-8, MCP-1, MIP-1b)secretion results with VISNE.

FIGS. 47A-47B depict the results from experiments investigating thecompensation between Alexa fluor 488 and 532 conjugate. FIG. 47A: Alexafluor 488 conjugated BSA was spotted onto poly-l-lysine glass slide. The488 channel signal and 532 channel signal showed good and stablecorrelation between each other (R²≈98%) and compensation equation can beextracted; FIG. 47B: correlation between real signal from 532 channeland crosstalk from 488 channel for Alexa fluor 532 conjugated BSA.

FIG. 48 is a set of graphs depicting the characterization of PMA (50ng/mL) differentiation of U937 monocyte for 48 hrs with macrophagemarkers CD11b (FL4) and CD14 (FL2).

FIG. 49 is a graph depicting the results of experiments comparing U937monocyte derived macrophage population cells protein secretion resultsfrom different substrates including 96 well plate, PDMS in 5:1, 10:1,20:1 ratio respectively. The results shows similar fold of change indifferent substrates for both high level secretion proteins like IL-8,MCP-1, IL-6 and low level secretion proteins like IL-1a, IL-3, IL-4.

FIG. 50 is a representative whole microchip heat map (from oneexperiment) that shows the secretion results from different cell numbersincluding 0 cells, single cell, 2 cells and multiple cells (≥3). Eachrow is a single cell and each column corresponds to a protein ofinterest. The signal from 0 cell microchambers can be used as threshold(average signal plus two times standard deviation) for positivesecretion.

FIGS. 51A-51B show a set of graphs demonstrating the comparison of U937monocyte derived macrophage single cell protein secretion results fromparallel two chips. FIG. 51A: the ratio of single cell signal vs zerocell signal are quite similar from these two chips. FIG. 51B: the ratiosof the single cell secretion signal from these two chips are very closeto 1.

FIG. 52 is a set of graphs depicting the correlation between U937macrophage protein secretion (IL-8, MCP-1, MIP-1b, MIF as examples) andcell numbers (0, 1, 2, 3 . . . ).

FIGS. 53A-53B depict the results of polyfunctionality analysis of U937macrophage (control and LPS stimulated). FIG. 53A presents a bar andline graph demonstrating the wide variety in the number of proteinssecreted. FIG. 53B depicts a set of pie graphs demonstrating thepercentage of cells secreting various ranges of proteins.

FIG. 54 depicts the comparison of VISNE and cluster results. Bothuntreated and LPS stimulated U937 single cells can be grouped into 3subpopulations with these two methods based on their protein secretionpatterns.

FIGS. 55A-55B show a set of graphs demonstrating U937 macrophage proteinsecretion dynamics between 0-48 hrs. FIG. 55A: control; FIG. 55B: LPSstimulated. The results shows different protein has different secretiondynamics at differing time points of analysis under selected stimulationconditions.

FIGS. 56A-56F depict the results of experiments demonstrating the U937derived macrophage protein secretion profile before (untreated) andafter 100 ng/mL LPS stimulation. FIG. 56A: All identical cells beforeand after stimulation. FIG. 56B: Identical single cells comparisonbefore and after stimulation. FIG. 56C-FIG. 56E: Scatterplots showingthe relation between IL-8 (before treatment and after) and IL-6, IL-10,TNF-a. FIG. 56F: the change of IL-8, MCP-1, Rantes, MMP-9 from the samecell before and after LPS stimulation.

FIG. 57 depicts a comparison of the single cell multiplexing array ofthe present invention (IsoPlexis) with CyTOF® Mass Cytometer system.

DETAILED DESCRIPTION

The present invention relates generally to a system, device, and methodfor high-throughput single-cell analysis. In one embodiment, theinvention is used to quantitatively detect the presence of a wide numberof compounds derived from single cells. For example, in one embodiment,the invention is used for the multiplexed detection of secretedcompounds from a single cell. In certain embodiments, the inventionallows for the multiplexed detection of secreted proteins, includingchemokines, cytokines, growth factors, antigens, and the like, fromsingle cells. The ability to analyze the secreted compounds from singlecells allows for assessment of the variability of cellular phenotypewithin a cell population. Further, the invention provides an effectivemechanism for identifying phenotypically rare and/or potential harmfulcell types within a population, whose activity would be otherwise hiddenin traditional population-based assays. In one embodiment, the device ofthe invention is a microchip comprising two independent components: (1)a high-density sub-nanoliter microwell array and (2) a substratecomprising a high-density capture agent array. In one embodiment, theinvention uses spatial and spectral encoding of compound capture.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “antibody” as used herein, refers to an immunoglobulinmolecule, which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoactive portions ofintact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as singlechain antibodies and humanized antibodies (Harlow et al., 1988; Houstonet al., 1988; Bird et al., 1988).

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

The term “polypeptide” as used herein is defined as a chain of aminoacid residues, usually having a defined sequence. As used herein theterm polypeptide is mutually inclusive of the terms “peptide” and“protein”.

By the term “specifically binds,” as used herein, is meant that acapture agent recognizes a specific compound, but does not substantiallyrecognizes or binds to other compounds in a sample. For example, anantibody that specifically binds to an antigen from one species may alsobind to that antigen from one or more species. But, such cross-speciesreactivity does not itself alter the classification of an antibody asspecific. In another example, an antibody that specifically binds to anantigen may also bind to different allelic forms of the antigen.However, such cross reactivity does not itself alter the classificationof an antibody as specific. In some instances, the terms “specificbinding” or “specifically binding,” can be used in reference to theinteraction of an antibody, a protein, or a peptide with a secondchemical species, to mean that the interaction is dependent upon thepresence of a particular structure (e.g., an antigenic determinant orepitope) on the chemical species; for example, an antibody recognizesand binds to a specific protein structure rather than to proteinsgenerally. If an antibody is specific for epitope “A”, the presence of amolecule containing epitope A (or free, unlabeled A), in a reactioncontaining labeled “A” and the antibody, will reduce the amount oflabeled A bound to the antibody.

As used herein, an “isolated feature” or “feature” refers to adistinguishable element found within the capture agent set describedherein. In certain embodiments, an isolated feature is a continuousline, non-continuous line, dot, square, triangle, or otherdistinguishable geometry, or combination of geometries. The geometry maybe any in which a capture agent set comprises reproducible numbers andarrangements of isolated features.

As used herein, an “interface” refers to a unit comprising one microwelland a capture agent set, described herein. An interface is formed whenthe microwell array and capture agent array of the invention arecontacted together in a consistent and repeated pattern.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention relates generally to a system, device, and methodfor high-throughput single-cell analysis. In certain embodiments, theinvention is used for the quantitative multiplexed detection of a widenumber of secreted compounds from single cells. The invention providesan effective mechanism for identifying single cell phenotypes within acell population, where single cell phenotype would be otherwise hiddenin traditional population-based assays. For example, the inventionallows for the identification of single cells that are phenotypicallyrare, or are of diagnostic value. In one embodiment, the inventionallows for the identification and quantification of single cellpolyfunctionality upon stimulation or challenge. In certain embodiments,the invention allows for the identification of potentially harmfulsingle cells within a cell population. The ability to analyze thesecreted compounds from single cells allows for assessment of thevariability of cellular phenotype within a cell population.

In one embodiment, the device of the invention is a microchip comprisingtwo independent components: (1) a high-density sub-nanoliter microwellarray and (2) a substrate comprising a high-density capture agent array.

The capture agent array is comprised of a plurality of capture agents,with each capture agent specifically recognizing a compound of interest.The capture agents are arranged in distinct isolated features along thesubstrate to provide spatial distinction between the various captureagents, herein described as “spatial encoding.” For example, in oneembodiment, the capture agent array is comprised of a plurality oflines, with each line comprised of one or more specific capture agent.In one embodiment, the capture agent array comprises repeats of isolatedfeature sets, where each set comprises all of the features needed toencompass all of the plurality of capture agents. As described elsewhereherein, the device of the invention allows for detection andquantification of 5-100 different compounds. Thus, the capture agentarray of the invention comprises 5-100 capture agents, each specific forone compound of interest. In certain embodiments, the capture agents ofthe invention specifically bind to a secreted protein of interest. Inone embodiment, the capture agents of the invention specifically bindone of 45 secreted proteins or antigens of interest, which are capturedwithin one microwell.

The multiplexing ability of the present invention allows for thedetection of 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20or more, 40 or more, 50 or more, 100 or more, and the like, compounds.For example, as is described elsewhere herein, the spatial and spectralencoding provided by the invention allows for the ability to detect verylarge number of different compounds.

It is noted herein that while the description presented herein describesa feature having one or more than one capture agent, it is not meantthat the feature has one molecule of the capture agent. Rather, askilled artisan will understand that a feature having an immobilizedcapture agent describes that the feature comprises some number orconcentration of a specific capture agent. Further, a feature havingthree capture agents is meant that the feature comprises some number orconcentration of three different specific capture agents.

The microwell array is comprised of a plurality of sub-nanoliter sizedmicrowells, each configured to house a single cell. Each microwellallows for constraint of a single cell while also, in certainembodiments, constraining the secreted compounds, secreted by the singlecell, to the microwell. As such, the microwell array of the inventionallows for analysis of the secreted proteins, specifically secreted bythe constrained single cell.

The device of the invention comprises the microwell array attached tothe capture agent array, to form interfaces between each microwell and acomplete set of capture agents disposed upon the capture agent array. Incertain embodiments, compounds secreted from a single cell, housed in amicrowell, bind specifically to a capture agent on the capture agentarray. Bound proteins are then visualized by, for example, use of agroup of labeled secondary capture agents in a sandwich ELISA.

In certain embodiments, bound compounds are visualized be the use of asecondary capture agent. In one embodiment, bound compounds arevisualized by use of a group of labeled secondary capture agents withone or more differently labeled secondary capture agents directed tobind to a particular line or feature, herein referred to as “spectralencoding,” as described elsewhere herein.

In one embodiment, the device of the invention allows for a multiplexedassay using the spatial position or shape of the plurality of isolatedfeatures to determine the presence or absence of each compound ofinterest. For example, observation of a detectable label (throughbinding of a labeled secondary detection agent), at a particular spatiallocation and/or within a distinguishable feature provides informationabout the presence of the particular compound.

In another embodiment, the device of the invention allows for amultiplexed spatial and spectral assay, where the distinguishablespatial feature as well as the color of selected labels at the featuredetermines the presence or absence of each compound of interest.

For example, in certain embodiments, each feature is comprised of morethan one capture agent, each of which corresponds to a distinct coloredlabel. For example, in one embodiment, the assay allows for thedetection of m×n compounds of interest, where m is equal to the numberof distinguishable isolated features, and n is equal to the number ofdetectable labels used per feature. Importantly, the assay does notrequire a unique label for each of the m×n compounds. Rather, thecombination of spatial features and colored labels provides only theneed for n different labels. For example, in one embodiment, threedifferent capture agents are positioned in each isolated feature, withthree differently labeled secondary capture agents used, eachdifferently labeled secondary capture agent corresponding to one of thethree positioned capture agents. In one embodiment, each set comprisesfifteen isolated features, which thereby allow the detection of 45(15×3) compounds per single cell.

The invention also provides a method for analyzing the secretome of asingle cell. The method comprises providing a microwell array andcontacting the array with a solution comprising a cell. The solutionspreads over the array such that the cell flows into a well of thearray. The method further comprises affixing a capture agent array tothe microwell array, such that the cell and solution within the well arein contact with a population of capture agents immobilized on thecapture agent array. Compounds (e.g. proteins) secreted from the singlecell are thus able to specifically bind to a capture agent located onthe capture agent array. In one embodiment, the method further comprisesadministering a group of secondary capture agents, which are tagged witha detectable label, to the capture agent array to form a detectablecomplex at a site of binding between a secreted compound and animmobilized capture agent.

The invention also provides a method of identifying the presence of acell with a specific phenotype within a sample obtained from a subject.As described elsewhere herein, the device of the invention allows forthe ability to detect the specific profile of secreted compounds fromindividual cells. Thus, the device and method of the invention allowsfor the identification of one or more cells whose profile is indicativeof a particular cellular phenotype. For example, in one embodiment, themethod comprises identifying a cell whose profile of secreted proteinsis indicative of a cancer cell, a malignant cancer cell, or the like. Inone embodiment, the method identifies subpopulations of individual cellswith specific characteristics.

Device

In one embodiment, the device of the invention comprises a microwellarray interfaced with a capture agent array. An image of an exemplarydevice is depicted in FIG. 21, while an exemplary schematic of thedevice and its components are shown in FIG. 1 and FIG. 22. As shown inFIG. 22, device 100 comprises microwell array 10 coupled to captureagent array 20. Device 100 couples individual microwells 11 with acomplete set 21 of capture agents to form a plurality of interfaces 101.Each interface 101 comprises an individual microwell 11 and a capturearray set 21, which is used to perform multiplexed detection of a widenumber of compounds present within microwell 11. Microwell array 10 andcapture agent array 20 are thus configured for precise coupling to allowfor high throughput detection and analysis from individual microwells.In one embodiment, the device comprises about 1,000-100,000 microwellscoupled to capture agent sets. Further description of microwell array 10and capture agent array 20 are provided below.

Microwell Array

In certain embodiments, the device of the invention comprises amicrowell array. The microwell array captures and constrains cells andtheir secreted compounds within the spatial limitations of each wellthereby preventing escape of the cells during implementation. Further,the microwell array allows cells to function normally in terms ofrelease of secreted compounds. In one embodiment, the cells remain alive(i.e. unfixed) and function normally within the microwell.

The microwell array of the invention comprises a plurality of individualmicrowells. In certain embodiments, the microwell array comprises ahigh-density array of individual microwells in order to provide a devicethat allows for high throughput analysis. The microwell array can be ofany shape or size suitable for the desired implementation of the device.In one embodiment, the microwell array is rectangular, having a definedlength and width.

In one embodiment, the microwell array is 0.5-10 cm in length. Inanother embodiment, the microwell array is 1-5 cm in length. In anotherembodiment, the microwell array is 2-3.5 cm in length.

In one embodiment, the microwell array is 0.5-10 cm in width. In anotherembodiment, the microwell array is 1-5 cm in width. In anotherembodiment, the microwell array is 2-3.5 cm in width.

In one embodiment, the microwell array comprises about 10-1,000,000individual microwells. In another embodiment, the microwell arraycomprises about 500-500,000 individual microwells. In anotherembodiment, the microwell array comprises about 100-100,000 individualmicrowells.

In one embodiment, the microwell array comprises microwells at a densityof about 200 microwells per cm² to about 20,000 microwells per cm².

As used herein, a microwell is a chamber that captures and constrainscells in an environment in which they remain alive and function. Eachmicrowell is configured to ensure reasonable survival of the cells. Forexample, cells within a microwell have a survival rate similar to thatexpected in an in vivo environment. Further, each microwell isconfigured to allow for normal functionality of captured cells duringthe length of the implementation of the device of the invention.

The dimensions of each microwell of the microwell array are designed toeffectively constrain cells and secreted compounds while promoting thenormal function and survival of constrained cells. The microwell isdesigned to cover the full area of one or more of the interfaced captureagent set. As such, the size and shape of each microwell is not limited,but rather can take any size and shape suitable for the cell type usedand multiplexing ability desired. In one embodiment, each microwell isrectangular, with a defined length, width and depth. Each microwellcaptures a sub-nanoliter volume of fluid, including, for example cellsuspension, physiological fluid, fluidic sample, possible reagents, andthe like.

In one embodiment, each microwell is 1-10,000 μm in length. In anotherembodiment, each microwell is 5-5,000 μm in length. In anotherembodiment, each microwell is 10-2,000 μm in length.

In one embodiment, each microwell is 1-1,000 μm in width. In anotherembodiment, each microwell is 5-500 μm in width. In another embodiment,each microwell is 10-100 μm in width.

In one embodiment, each microwell is 1-1,000 μm in depth. In anotherembodiment, each microwell is 5-500 μm in depth. In another embodiment,each microwell is 10-100 μm in depth.

In one embodiment, each microwell is a high aspect ratio rectangularwell. For example, in one embodiment, each microwell is about 1.8 mmlong and about 20 μm wide.

In one embodiment, each microwell is about 10-100 μm in width, 20-200 μmin depth, and 100-2,000 μm in length.

In certain embodiments, individual microwells are spaced laterally andlongitudinally in an array of rows and/or columns. In one embodiment,individual microwells are regularly spaced at about 10-100 μm inseparation.

Fabrication of the microwell array may be done making use of anysuitable method or methods necessary to construct the array describedherein. For example, in one embodiment, the microwell array isfabricated by negative photolithography wafer molding and subsequentsoft-polymer (PDMS, Sylgard 184) using standard procedures for laydown.In one embodiment, soft lithography techniques are used for elastomerstamping and molding of the array.

In one embodiment, the microwell array is optically transparent to allowfor imaging of the array or of a cell or cells within the array. Incertain embodiments of the invention, imaging of the array is desiredfor cell-counting, microwell alignment, and/or determination of cellularlocalization within a microwell.

In one embodiment, the microwell array is manufactured and configuredfor a single use. In another embodiment, the microwell array ismanufactured and configured to be reusable. For example, in certainembodiments, the microwell array is reusable after a standard washprocedure using water, saline, buffers, or a combination thereof. Incertain embodiments, the microwell array is suitable to be sterilized,for example by use of an autoclave or UV irradiation.

In one embodiment the microwell is functionalized or otherwisemanipulated for the purposes of cell tethering or other immobilization.

Each microwell is configured to capture and constrain an individual cellor cells over the area of the accompanying capture agent array. Forexample, each microwell may be specifically designed such that themicrowell contains a desired number of cells of a particular cell type.In certain embodiments, each microwell contains 1, 2, 3, 5, 10, 15, 20,50, or 100 cells. In a particular embodiment, each microwell isconfigured to contain a single cell. As used herein, a single cell isdefined as an individual cell of any cell type or cell line. In certainembodiments, the cell is a secretory cell. In other embodiments, thecell is a non-secretory cell. In a preferred embodiment, the single cellremains alive for some, most, or all of the implementation time of thedevice. In a more preferred embodiment, the single cell remains aliveduring the entire duration of the implementation of the device. Forexample, in certain embodiments, the device is used to assay the profileof secreted proteins from a single cell during a 24 hour period. It ispreferred that the microwell allows for survival of a single cell forthe 24 hour period.

In one embodiment, the microwell array is configured to be compatiblewith the capture agent array, such that when the microwell array isaffixed to the capture agent array, each individual microwell, and cellsand proteins contained therein, are in contact with at least one fullset of capture agent features (e.g. lines, dots, etc) to ensure that allof the plurality of capture agents are accessible to the contents of themicrowell.

Capture Agent Array

In certain embodiments, the device of the invention comprises a captureagent array. The capture agent array comprises a plurality of captureagent sets, where each capture agent set comprises all of the pluralityof capture agents desired for the multiplexed detection of compounds. Asdescribed elsewhere herein, each set is configured to be accessible tothe contents of a single microwell.

In certain embodiments, the capture agent array comprises repeats ofcapture agent sets. In one embodiment, each set is spatially distinctfrom all other sets. In one embodiment, each set is separated by about10-100 μm.

In one embodiment, the capture agent array comprises 10-1,000,000individual capture agent sets. In another embodiment, the capture agentarray comprises about 500-500,000 individual capture agent sets. Inanother embodiment, the capture agent array comprises about 100-100,000individual capture agent sets.

Each set is sized and shaped to correspond with one or more microwells,as described above. For example, in one embodiment each set correspondsto a single microwell. Assembly of the device by contacting themicrowell array with the capture agent array thereby forms a pluralityof interfaces, with each interface comprising a microwell and a set. Thedevice is only limited in that the contents of each microwell must beaccessible to all the isolated features comprised in a set, as furtherdescribed elsewhere herein. In certain embodiments, each set isconfigured based upon the size of multiplexing desired. Thus, in oneembodiment, each set is rectangular having with a defined length andwidth

In one embodiment, each set is 1-10,000 μm in length. In anotherembodiment, each set is 5-5,000 μm in length. In another embodiment,each set is 10-2,000 μm in length.

In one embodiment, each set is 1-1,000 μm in width. In anotherembodiment, each set is 5-500 μm in width. In another embodiment, eachset is 10-100 μm in width.

As shown in FIG. 23A, in certain embodiments, each set 21 comprises aplurality of spatially isolated features 22 that comprise one or moredifferent immobilized capture agents. Device 10 is assembled such aninterface 101 is formed between each set 21 and each microwell 11, suchthat the contents of each microwell 11 are accessible to each of theplurality of features 22. For example, in one embodiment, set 21comprises a plurality of features 22, where each feature 22 is a line,where each line comprises one or more immobilized capture agents.Features 22 are not limited to any particular size or shape. Forexample, in one embodiment, features 22 are straight lines. In anotherembodiment, features 22 are zig-zag lines (as depicted in FIG. 23). Eachfeature 22 comprises one or more immobilized capture agents for thedetection of a compound of interest. For example, in one embodiment, set21 comprises twenty individual features 22, with each feature comprisinga specific capture agent to detect one of twenty compounds of interest.In one embodiment, the capture agent immobilized on one feature 22 isnot comprised in another feature 22. Thus, detecting compound binding ata particular feature 22, as described elsewhere herein, providesinformation as to the presence of a particular compound of interest.

FIG. 23B, depicts an alternative embodiment of a comprising a pluralityof isolated features 122. In this embodiment, each feature 122 comprisesa distinctive shape or geometry. Each feature 122 comprises one or moreimmobilized capture agents immobilized in the particular shape offeature 122. Micropatterning technology can be used to fabricate featuresize and shape with very high resolution. Exemplary techniques includemicroinject printing, microcontact printing, dip-pen lithography,microchannel-guided flow patterning, and the like. However, the deviceis not limited to any particular method of immobilizing capture agentsinto isolated features. For example, FIG. 24, depicts exemplary captureagent arrays, comprising capture agents arranged as squares or diamondfeatures. This array is fabricated by cross-flow patterning techniqueand can also be fabricated by micro-scale printing techniques such asmicro spotting and inject printing.

Isolated features allow for the determination of which compound (orcombination of compounds) are present within each microwell. Forexample, the spatial location of the feature, the shape of the feature,and/or the combination thereof is used to identify which compound ofinterest (or combination of compounds of interest) bound to its specificcapture agent immobilized on the feature. Further, as each setcorresponds to an individual microwell, the spatial location of acapture agent set on the capture agent array allows for thedetermination of within which individual microwell the compound (orcombination of compounds) were present. This therefore allows fordetermining an individualized profile stemming from the contents of eachmicrowell. In certain embodiments, the isolated features are repeatingand at least one set of isolated features in full in contained permicrowell. In one embodiment, two or more sets may be contained in eachmicrowell, for example as a control.

In one embodiment, detection of the bound compound of interest is madewith an ELISA-based immunoassay, as described elsewhere herein.Importantly, the use of distinguishable isolated features (spatiallydistinguishable and/or distinguishable by shape or geometry) allows formultiplexing for a large number of compounds from single cells, as aunique secondary antibody label is not needed for each compound. Thistype of “spatial encoding” allows for the detection of the relevantsecreted compounds for each microwell from its observed binding to aspatial location within the isolated feature set interfaced with thespecific microwell, rather than a specific label on each secondarycapture agent specific to the compound, as in traditional ELISA. Forexample, traditional assays require a 1:1 compound to detectable labelrelationship in order to correlate the label to the particular compoundof interest. As described herein, the use of isolated features allowsthat each compound be associated with the same detectable label (or atleast same group of labels used in spectral assays described elsewhereherein).

As described elsewhere herein, each specific capture agent specificallybinds to a compound (protein, antigen, receptor, nucleic acid, etc) ofinterest. Each isolated feature has a defined spatial location and/orshape within the capture agent set, thereby allowing for easy andeffective determination of whether or not a specific compound ofinterest is present in the corresponding microwell. For example, in oneembodiment, each isolated feature is spaced at about 2-25 μm from oneanother. In one embodiment, each set comprises at least one of theplurality of isolated features. That is, each isolated feature isrepresented at least once in each set. In some embodiments, one or moreisolated features are repeated a multitude of times within eachinterface. For example, in one embodiment, an interface may comprise asingle well interfaced with multiple sets.

In one embodiment, each set comprises a total of about 5-100 differentcapture agents. In another embodiment, each set comprises a total ofabout 10-75 different capture agents. In another embodiment, each setcomprises a total of about 20-50 different of capture agents. Forexample, in one embodiment, the invention provides for a 45-plexeddetection of compounds, wherein each set comprises 45 different captureagents.

In one embodiment, each set comprises about 5-100 isolated features. Inanother embodiment, each set comprises about 10-50 isolated features. Inanother embodiment, each set comprises about 20-30 isolated features.

In certain embodiments, isolated features are continuous over multiplecapture agent sets. For example, in some fabrication techniques it maybe easier to fabricate continuous lines or shapes that would span over aplurality of sets. Contacting the capture agent array with the microwellarray then breaks the continuous features into individual setscorresponding to individual microwells. In another embodiment, thecapture array is fabricated such that isolated features arenon-continuous, and are thus placed only at locations that willcorrespond to the interface between a microwell and capture agent set.

In certain embodiments, each isolated feature (e.g. line, shape, etc)comprises a single specific capture agent. In another embodiment, eachisolated feature comprises 2, 3, 5, 10, or more different captureagents. In these embodiments, specific binding to a particular captureagent within the feature is determined by use of different detectablelabels on a second set of capture agents used in an ELISA-based assay,with each label corresponding to a particular capture agent, termedherein as “spectral encoding.”

For example, in one embodiment, the array comprises at least one setcomprising fifteen features (e.g. lines, dots, or the like). In oneembodiment, the first feature comprises one or more molecules of a firstcapture agent, the second feature comprises one or more molecules of asecond capture agent, the third feature comprises one or more moleculesof a third capture agent, and so on. This is repeated until thefifteenth feature, which comprises one or more molecules of a fifteenthcapture agent.

In one embodiment, each feature comprises more than one capture agent.For example, in one embodiment, the first feature comprises one or moremolecules of a first capture agent, one or more molecules of a secondcapture agent, and one or more molecules of a third capture agent; whilethe second feature comprises one or more molecules of a fourth captureagent, one or more molecules of a fifth capture agent, and one or moremolecules of a sixth capture agent. This is repeated until the fifteenthfeature, which comprises one or more molecules of the forty-thirdcapture agent, one or more molecules of the forty-fourth capture agent,and one or more of the forty-fifth capture agent. In this embodiment,each capture agent within a feature is distinguishable by the color of asecondary capture agent targeted to the capture agent, defined asspectral encoding as described elsewhere herein.

For example, in one embodiment, an isolated feature comprises threedifferent capture agents, each specific for a different compound ofinterest. Determination of which of the three compounds of interest (orcombination of compounds of interest) is found in the microwell is doneby applying a second set of three different capture agents, one labeledwith a red label, one with a green label, and one with a blue label, tothe immobilized capture agents in an ELISA based reaction to produce adetectable complex at the feature. Detecting the presence of a red,green, and blue label at the spatial location of each isolated featurein the capture agent arrays thus allows for determination of which ofthe three compounds (or combination of three compounds) of interest ispresent per said isolated feature. Scaling this principle over all theisolated features present in the capture agent set allows for themultiplexed detection of a very large number of compounds. For example,devices have been configured and demonstrated to detect for up to 60different compounds.

In certain embodiments, the invention provides spatial and spectralmultiplexing of compounds of interest. For example, in one embodiment,the ability to multiplex is partially defined by the number of spatiallydistinguishable isolated features per capture agent set. In oneembodiment, each isolated feature is about 5-50 μm in width. In oneembodiment, each isolated feature is about 25-30 μm in width. In oneembodiment each isolated feature is about 500-2000 μm in length. In oneembodiment, each isolated feature is separated by about 5-50 μm. In oneembodiment, each isolated feature is separated by about 25-30 μm. In oneembodiment, each isolated feature is about 25 μm in width and separatedby about 25 μm from the next feature, thereby giving a pitch size ofabout 50 μm. The present invention demonstrates the ability to producevery small spatially distinguishable isolated features to therebyincrease the spatial multiplexing ability of the device.

In one embodiment, the ability to multiplex is partially defined by thenumber of spectrally distinct labels used such that each isolatedfeature can contain more than one capture agent, thus allowing fordetection of more than one type of compound of interest per isolatedfeature. For example, in one embodiment, the capture array set comprises15 spatially distinct features, each feature comprising 3 differentcapture agents, distinguishable through use of 3 different ELISA-basedsecondary capture agents conjugated to different detectable labels. Thisconfiguration therefore allows for 45-plexed detection.

The capture agent array performs a multiplexed highly-quantitativeanalysis of secretions per single cell by coupling to the single-cellisolating microwells. The repeating sets serve to allow each microwellto capture a full detection range of 5-100 compounds per single cellwithout significant deviation in accuracy of capture between eachmicrowell due to the uniformity of design.

Patterning of capture agents at isolated features and sets is achievedthrough any suitable technique. For example, Capture agent patterningcan be achieved at high accuracy through inkjet printing, fine printspotting, flow patterning on a functionalized substrate, contactprinting on functionalized glass substrate, or microprinting usingepoxy-coated glass substrate or poly-amine glass substrate with printingneedles or strips at 2-30 μm feature resolution. In certain embodiments,the functionalized substrate comprises a poly-L-lysine coated substrate.However, any functionalized substrate that provides the physical and/orchemical affinity to immobilize the capture agents to a high degree ofreproducibility with limited cross-reactivity of the capture agents inthe array of the invention may be used.

As described above, each capture agent set comprises a plurality ofcapture agents, where each capture agent specifically binds to acompound of interest being assayed in the desired implementation.Exemplary compounds of interest, include, but are not limited toproteins, peptides, antibodies, enzymes, surface receptors, nucleicacids, peptide fragments, cytokines, growth factors, hormones, and thelike. In certain embodiments, the compound or compounds of interest arecompounds known or thought to be secreted by the cell or cells containedwith the microwell of the device. However, the invention is not limitedto detection of secreted compounds, but rather any compound that isaccessible to the capture agent array. For example, a compound ofinterest can include one that is accessible upon lysis of the cell,either through the death of the cell or following user manipulation.

Capture agents include, but are not limited to, antibodies, antibodyfragments, proteins, peptides, and nucleic acids. In certainembodiments, capture agents have a capture affinity of about 1 pM toabout 150 pM to their target compound. As described elsewhere herein,detection of the binding between the immobilized capture agent on thearray and its target compounds is performed through ELISA-based couplingusing a second group of capture agents, where each of the second groupof capture agents specifically binds to a capture agent-compoundcomplex. In certain embodiments, each member of the second group ofcapture agents is labeled with a detectable label, including, but notlimited to, a dye or fluorophore. The secondary capture agents maycomprise antibodies, antibody fragments, proteins, peptides, and nucleicacids. In some embodiments, a secondary capture agent specifically bindsan immobilized capture agent of the capture agent array. In anotherembodiment, a secondary capture agent, such as a detection antibody,specifically binds the compound of interest.

In one embodiment, the capture agent of the invention comprises apeptide. In certain embodiments, the peptide capture agent specificallybinds to a compound of interest, for example a secreted compound ofinterest.

The peptide of the present invention may be made using chemical methods.For example, peptides can be synthesized by solid phase techniques(Roberge J Y et al (1995) Science 269: 202-204), cleaved from the resin,and purified by preparative high performance liquid chromatography.Automated synthesis may be achieved, for example, using the ABI 431 APeptide Synthesizer (Perkin Elmer) in accordance with the instructionsprovided by the manufacturer.

The peptide may alternatively be made by recombinant means or bycleavage from a longer polypeptide. The composition of a peptide may beconfirmed by amino acid analysis or sequencing.

The variants of the peptides according to the present invention may be(i) one in which one or more of the amino acid residues are substitutedwith a conserved or non-conserved amino acid residue (preferably aconserved amino acid residue) and such substituted amino acid residuemay or may not be one encoded by the genetic code, (ii) one in whichthere are one or more modified amino acid residues, e.g., residues thatare modified by the attachment of substituent groups, (iii) one in whichthe peptide is an alternative splice variant of the peptide of thepresent invention, (iv) fragments of the peptides and/or (v) one inwhich the peptide is fused with another peptide, such as a leader orsecretory sequence or a sequence which is employed for purification (forexample, His-tag) or for detection (for example, Sv5 epitope tag). Thefragments include peptides generated via proteolytic cleavage (includingmulti-site proteolysis) of an original sequence. Variants may bepost-translationally, or chemically modified. Such variants are deemedto be within the scope of those skilled in the art from the teachingherein.

As known in the art the “similarity” between two peptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one peptide to a sequence of a second peptide. Variantsare defined to include peptide sequences different from the originalsequence, preferably different from the original sequence in less than40% of residues per segment of interest, more preferably different fromthe original sequence in less than 25% of residues per segment ofinterest, more preferably different by less than 10% of residues persegment of interest, most preferably different from the original proteinsequence in just a few residues per segment of interest and at the sametime sufficiently homologous to the original sequence to preserve thefunctionality of the original sequence. The present invention includesamino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%,78%, 80%, 90%, or 95% similar or identical to the original amino acidsequence. The degree of identity between two peptides is determinedusing computer algorithms and methods that are widely known for thepersons skilled in the art. The identity between two amino acidsequences is preferably determined by using the BLASTP algorithm [BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894,Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

The peptides of the invention can be post-translationally modified. Forexample, post-translational modifications that fall within the scope ofthe present invention include signal peptide cleavage, glycosylation,acetylation, isoprenylation, proteolysis, myristoylation, proteinfolding and proteolytic processing, etc. Some modifications orprocessing events require introduction of additional biologicalmachinery. For example, processing events, such as signal peptidecleavage and core glycosylation, are examined by adding caninemicrosomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489)to a standard translation reaction.

The peptides of the invention may include unnatural amino acids formedby post-translational modification or by introducing unnatural aminoacids during translation.

In one embodiment, the capture agent of the invention comprises anantibody, or antibody fragment. In certain embodiments, the antibodycapture agent specifically binds to a compound of interest, for examplea secreted compound of interest. Such antibodies include polyclonalantibodies, monoclonal antibodies, Fab and single chain Fv (scFv)fragments thereof, bispecific antibodies, heteroconjugates, human andhumanized antibodies.

Such antibodies may be produced in a variety of ways, includinghybridoma cultures, recombinant expression in bacteria or mammalian cellcultures, and recombinant expression in transgenic animals. The choiceof manufacturing methodology depends on several factors including theantibody structure desired, the importance of carbohydrate moieties onthe antibodies, ease of culturing and purification, and cost. Manydifferent antibody structures may be generated using standard expressiontechnology, including full-length antibodies, antibody fragments, suchas Fab and Fv fragments, as well as chimeric antibodies comprisingcomponents from different species. Antibody fragments of small size,such as Fab and Fv fragments, having no effector functions and limitedpharmokinetic activity may be generated in a bacterial expressionsystem. Single chain Fv fragments show low immunogenicity.

In one embodiment, the capture agent of the invention comprises anisolated nucleic acid, including for example a DNA oligonucleotide and aRNA oligonucleotide. In certain embodiments, the nucleic acid captureagent specifically binds to a compound of interest, for example asecreted compound of interest. For example, in one embodiment, thenucleic acid comprises a nucleotide sequence that specifically binds toa compound of interest. For example, in one embodiment, the nucleic acidis complementary to a secreted nucleic acid of interest.

The nucleotide sequences of a nucleic acid capture agent canalternatively comprise sequence variations with respect to the originalnucleotide sequences, for example, substitutions, insertions and/ordeletions of one or more nucleotides, with the condition that theresulting nucleic acid functions as the original and specifically bindsto the compound of interest.

In the sense used in this description, a nucleotide sequence is“substantially homologous” to any of the nucleotide sequences describeherein when its nucleotide sequence has a degree of identity withrespect to the nucleotide sequence of at least 60%, advantageously of atleast 70%, preferably of at least 85%, and more preferably of at least95%. Other examples of possible modifications include the insertion ofone or more nucleotides in the sequence, the addition of one or morenucleotides in any of the ends of the sequence, or the deletion of oneor more nucleotides in any end or inside the sequence. The degree ofidentity between two polynucleotides is determined using computeralgorithms and methods that are widely known for the persons skilled inthe art. The identity between two amino acid sequences is preferablydetermined by using the BLASTN algorithm [BLAST Manual, Altschul, S., etal., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol.Biol. 215: 403-410 (1990)].

System for Multiplexed Detection

In one embodiment, the present invention includes a system formultiplexed detection of compounds secreted by a single cell. In certainembodiments, the system comprises the device described in detailelsewhere herein. That is, in one embodiment the system comprises adevice comprising a microwell array and a capture agent array, asdescribed above.

In one embodiment, the device of the system comprises a capture agentarray having a plurality of sets, each set comprising a plurality ofisolated features, wherein each feature comprises one or moreimmobilized capture agents that bind to a specific compound of interest.

In one embodiment, the system comprises a group of secondary captureagents which are administered to the device following incubation ofpopulation of single cells with the immobilized capture agents of thecapture agent array. The secondary capture agent specifically binds toan immobilized capture agent, the compound of interest, or to animmobilized capture agent-compound complex. That is, the secondarycapture agent is used in a sandwich-type assay, and in certainembodiments aid in the detection of a compound bound to the captureagent array.

In certain embodiments, the secondary capture agents compriseantibodies, antibody fragments, proteins, peptides, and nucleic acids.In one embodiment, the secondary capture agents are labeled with adetectable label. Secondary capture agents, specific for binding animmobilized capture agent-compound complex, can be labeled with anydetectable label, including but not limited to, fluorescent labels,radioactive labels, ferromagnetic labels, paramagnetic labels,luminescent labels, electrochemiluminescent labels, phosphorescentlabels, chromatic labels, and the like. Non-limiting examples offluorescent labels include, green fluorescent protein (GFP), cyanfluorescent protein (CFP), yellow fluorescent protein (YFP), redfluorescent protein (RFP), orange fluorescent protein (OFP), eGFP,mCherry, hrGFP, hrGFPII, streptavidin APC, Alexa 488, Alexa 532, Alexa594, and the like. Fluorescent labels may also be photoconvertable suchas for example kindling red fluorescent protein (KFP-red), PS-CFP2,Dendra2, CoralHue Kaede and CoralHue Kikume.

In one embodiment, the secondary capture agent is labeled with a tagregion, including for example a peptide or other epitope. For example,in one embodiment, the secondary capture agent is labeled with biotin(i.e. biotinylated), such that the use of a fluorescently labeledstreptavidin, or other detectably labeled streptavidin, is used tovisualize the biotinylated secondary capture agent.

In one embodiment, all secondary capture agents are labeled with thesame detectable label. In another embodiment, the secondary captureagents are differently labeled, in order to differentiate the binding ofone secondary capture agent from another. In one embodiment, eachcapture agent immobilized on the capture agent array is assigned acorresponding labeled ELISA-detection secondary capture agent, such thateach capture agent at a given feature has a distinct correspondinglabel. In certain embodiments, the plurality of labels corresponding tothe plurality of immobilized capture agents within a given feature donot exhibit any cross-reactivity during the spectral encodingembodiments presented herein.

For example, in one embodiment, the group of secondary capture agentscomprise one or more subgroups, with each subgroup having a particulardetectable label. The system is thus devised such that a secondarycapture agent from each subgroup is configured to bind to a singlespecific immobilized capture agent-compound complex formed at a specificfeature. Thus, if, for example, all compounds were present in a givenmicrowell, all of the different detectable labels would be observed ateach feature. The differentially labeled secondary capture agents allowsfor the spectral encoding of the presence of a given compound. That is,the combination of the spatial location of the label and the precisetype (i.e. color) of the label, identifies the compound that is present.

In one embodiment, the system comprises a detector for detecting theidentity and location of each detectable label. The detector may be anysuitable detector that is capable of detecting each label, including,but not limited to a fluorescent microscope, fluorescent detector, orfluorescent scanner.

In one embodiment, the system comprises a computing device. Thecomputing device may include a desktop computer, laptop computer,tablet, smartphone or other device and includes a software platform forcontrol of the system components, display of raw data, and analysis ofacquired data. The computing devices may include at least one processor,standard input and output devices, as well as all hardware and softwaretypically found on computing devices for storing data and runningprograms, and for sending and receiving data over a network.

In certain embodiments, the system of the invention comprises hardwareand software which detect and quantify detection signals from the array.The signals may be quantified using any suitable analysis softwarepackage, or using custom made analysis algorithms. Exemplary analysesand graphical output of data are presented elsewhere herein.

Methods of Multiplexed Detection

The present invention provides methods of simultaneously detecting alarge number of compounds of interest in a small sample. For example, inone embodiment, the invention allows multiplexed detection of proteinsfrom a single cell housed in a microwell, as described elsewhere herein.The method allows for the determination of individualized profiles fromsingle cells, which in certain embodiments is preferred over profilesfrom a population of cells. The ability to discriminate profiles fromindividual cells can aid in the determination of a particular cellularphenotype, hiding within a total population. This would, for example, beuseful in detecting a cancerous cell, malignant cell, or metastasizingcell, in a tissue sample, that would otherwise be difficult or nearimpossible to detect. In certain embodiments, the method provides forthe ability to calculate an average single cell parameter(s) thatprovide more information than population based assays. Such analysis canbe used, for example, to identify sub-populations and/or groupedphenotypes. Isolating these subpopulations of individual cells, groupedby single cell multi-plexed parameters, can be valuable for isolatingimportant active groups of malignant cancer cells and responsive immunecells in a tissue sample. The multiplexing ability of the presentinvention therefore allows for the isolation or identification ofphenotypic subtypes. For example, the method identifies the relativeamount of a given phenotype within a population. Further, such analysiscan provide, but is not limited to, the quantification ofcross-correlation between secreted compounds or cellular products, thecreation of population or whole chip compound distribution statistics,and the evaluation of polyfunctionality expressed on the single celllevel over the population tested.

In one embodiment, the method comprises loading a single cell into amicrowell described herein. The device of the present invention allowsfor microwell loading that does not require active fluidics (i.e. pumps,pressurized flow, etc) or external force manipulation of live cells.Rather, the method comprises loading a single cell into a microwellusing gravity alone. This allows for a method in which single cells areisolated and profiled in live conditions without extensive manipulation,which may be time consuming and/or expensive.

The cells profiled by way of the method of the invention may be of anysuitable cell type. Applicable cells for consideration include bothadherent and non-adherent cells, primary cells and immortalized celllines, cells from organ tissue, and cells grown ex vivo. In certainembodiments, a cell is administered to the microwell in the form of asolution comprising the cell. For example, the solution can be asingle-cell suspension, a media cell suspension, or a physiologicalfluid naturally comprising the cell. The cell may be from a cell line orisolated from a subject, including a human. In one embodiment, thesolution is derived from tissue isolated from a subject. For example,the solution is derived from homogenized tissue.

In one embodiment, the method for loading a cell into the microwellcomprises adding a solution comprising a cell directly over a wettedsurface of the microwell array. For example, the surface of themicrowell array may be wetted with water, saline, buffer, or a suitablecell culture medium. In one embodiment, addition is made in a singlemotion over approximately the middle of the microwell array at leastabout 0.1 mm above the surface. Addition of the solution to the surfacemay be done under any standard pipetting or liquid transfer method knownin the art for standard cell culture preparation. The volume of thesolution added to the microwell array surface is minimal compared toavailable multiplex analysis tools. In one embodiment, the methodcomprises administering about 1-500 μL to the surface. The number ofcells within the solution is dependent upon the availability of thecells and the desired throughput. For example, the solution can compriseas low as about 10³, 10², or 10¹ cells. Upon addition of solution to themicrowell array surface, the cells of the solution fall into eachindividual microwell by gravity alone and exhibit a Poisson distributionof cells per well. In certain embodiments, wells with zero cells ormultiple cells can be later eliminated from analysis, or included forcontrol and/or background signal processing.

In certain embodiments, the solution comprises one or more componentsthat promote the survival and/or normal function of the cell duringimplementation. For example, the solution can comprise growth factors,hormones, proteins, enzymes, small molecules, antimicrobials, and thelike typically used in cell culture. In some embodiments, the solutioncomprises suitable cell culture medium. In some embodiments, thesolution comprises a test agent or test compound whose effects aredesired to be assayed during implementation of the invention. Forexample, the test agent can comprise a small molecule, protein, nucleicacid, peptide, or the like which may or may not have an effect on thedetected profile. For example, the agent may or may not increase thesecretion of one or more compounds of interest or decrease the secretionof one or more compounds of interest.

The method further comprises contacting the capture agent array with themicrowell array. Creating the proper interface between the microwellarray and capture agent array, with the alignment of individualmicrowells with their corresponding capture array sets, is performed, inone embodiment, by a pressurized clamp mechanism using an integrateddevice housing. It is noted herein, however, that no precise alignmentof the microwell array and capture agent array is necessary. Forexample, the uniformity of the arrays allow for a loose alignment thatdoes not necessarily require a specific microwell to align with aspecific set on the capture array. Further, in certain embodiments,automated analyses conducted post-experiment performs signal matching,which can overcome non-precise alignment. However, the present method isnot limited to any particular method of forming this interface. Forexample, permanent and non-permanent adhesives, screws, clamps, and thelike may also be used.

This loading method is solely dependent upon the size of the individualcells and size of microwells, which both dictate the average number ofsingle cells per microwell. In some embodiments, the loading proceduredescribed herein ensures the constraint of about 0 to 20 cells permicrowell. The overall distribution of the number of cells per wellapproximates a Poisson distribution.

In one embodiment, the cells are constrained within the device for adesired period of time over single or multiple time points. In oneembodiment, the method comprises constraining the cells within thedevice for about 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2hours, 4 hours, 8 hours, 16 hours, 1 day, 2 day, 4 days, 1 week, 2weeks, 1 month, one year, or more.

In one embodiment, the method comprises acquiring images recording thenumber and location of cells within each microwells. In certainembodiments, this allows for the determination of whether a determinedprofile was produced by a single cell or rather by a population ofsingle cells. Image acquisition may be carried out by any method knownin the art.

In one embodiment, after the desired time course as elapsed, the deviceis disassembled to remove the capture agent array. The capture agentarray is then subjected to known ELISA immunoassay procedures to producedetectable complexes at the sites of compound binding. For example, inone embodiment, a plurality of secondary capture agents, each specificfor a different immobilized capture agent-compound complex, as describedelsewhere herein, is applied to the surface of the capture agent arrayunder suitable conditions to promote specific binding. As detailedelsewhere herein, each secondary capture agent is labeled with adetectable label, for example, a fluorescent label. In certainembodiments, each of the plurality of secondary capture agents are alllabeled with the same label. In other embodiments, secondary captureagents are labeled with different labels to spectrally discriminatebetween complexes that may have been formed at the same spatiallocation.

In one embodiment, following application of the group of secondarycapture agents, the capture agent array is imaged for the presence ofone or more detectable labels at distinct spatial locations. FIG. 25depicts an exemplary read-out illustrating (1) a bright-field image of agroup of microwells used to detect the presence and/or quantity of cellsper microwell, (2) a fluorescent image to detect the presence of adetectable label at specific spatial locations, with each locationcorresponding to a particular compound of interest, and (3) an overlayof the bright field and fluorescent image.

Imaging of the capture agent array may be done by any suitable methodknown in the art. For example, in certain embodiments, imaging of thecapture agent array comprises use of a fluorescent microscope,fluorescent detector, and/or fluorescent scanner. Image analysis,including the determination of which spatial features of each captureagent set have a detectable label, is then used to construct amultiplexed profile from each set, which thus corresponds to a profilefrom each microwell. In certain embodiments where a plurality ofdetectable labels is used, images may be color combined prior toanalysis.

Methods of Single Cell Phenotype Determination

The present invention provides a method to elucidate a single cellprofile. For example, in one embodiment, the invention provides a methodto determine a proteomic profile from a single cell. In anotherembodiment, the invention provides a method to determine a genomicprofile from a single cell. In another embodiment, the inventionprovides a method to determine a secretomic (i.e. secreted proteins andcompounds) from a single cell. In another embodiment, the inventionprovides a method to determine a combined proteomic, genomic, and/orsecretomic profile from a single cell. Further the method provides ahigh throughput method of determining the profiles for a large quantityof single cells simultaneously.

In one embodiment, the method of the invention is used to evaluateheterogeneity or multifunctionality of immune cells. Immune cells havean essential role in prevention and protection against a variety ofinfections (Seder et al., 2008, Nature Reviews Immunology, 8: 247).Despite the rapid development of immune cell phenotyping using thepowerful flow cytometry technology, it remains difficult to fullydissect the functions of different phenotypes due in part to theextremely high level of cellular heterogeneity including in T cells andmonocyte/macrophage (Gordon et al., 2005, Nature Reviews Immunology, 5:953-964). Moreover, such heterogeneity exists not only at the level ofphenotype but also at the level of cell behavior as reflected by thediverse effector functions and activated states (Seder et al., 2008,Nature Reviews Immunology, 8: 247). An immune cell often displays anumber of functions, termed multifunctionality, and the combinations ofmultiple functions determines the immunobiology of a single cell and the“quality” of this cell against a given infection. In a simplified lineardifferentiation model of the T_(H)1 cells, both phenotypic andfunctional heterogeneity have been observed in term of CD4⁺ T-cellcytokine responses. It is noted that these cells display differenteffector functions (cytokine profiles) at different stages and suchfunctional patterns dynamically evolve with time. It was also found thatcells with multiple functions serve best as the memory CD4⁺ T cells withpotent effector potential. The truth is likely far more complex thanthis linear differentiation model, and over 40 effector functions areassociated with helper T cells. The importance of multifunctionality isfurther exemplified by the observation that non-progressor AIDS patientsdeveloped a repertoire of HIV-specific T cells that secret a greaternumber of different cytokines as compared to the progressor patients.However, there are no technologies available to assess the full spectrumof immune effector functions at the single cell level, and the cellularimmunobiology of these dynamically evolving cells remain poorlyunderstood. In certain embodiments, the present method allows for theevaluation of the heterogeneity of immune cells by detecting thesecretome of one or more single immune cells, by use of the presentlydescribed multiplexed system. For example, the secretome of singleimmune cells can be evaluated when left untreated, or when stimulatedwith one or more test compounds. This can thus be used to evaluate thequality of a subject's immune response.

The singular term “cancer” is never one kind of disease, but deceivinglyencompasses a large number of heterogeneous disease states, which makesit impossible to completely treat cancer using a generic approach. Thisis due in part to the significant intratumoral heterogeneity (Furnari etal., 2007, Genes Dev, 21: 2683-2710). Moreover, such heterogeneity is soprofound that it exists at the single cell level within a tumormicroenvironment. For example, in human brain tumor glioblastomamultiforme, there are never just glioma cells, but also other key celltypes such as tumor initiating immune cells (Bao et al., 2006, CancerResearch, 66: 7843-7848; Singh et al., 2004, Nature, 432: 396-401),neural/glial progenitor cells, neurons, astrocytes, oligodendrocytes andthe brain-resident immune cells —microglia. Such a remarkableheterogeneity of gliomblastoma microenvironment and the lineagerelationships of different cells within a solid human tumor can be animportant determinant of tumor cell competencies. Individual GBMs canharbor a series of phenotypically distinct self-renewing cell types thatpromote a range of tumor growth patterns (Chen et al., 2010, CancerCell, 17: 362-375). Thus, it is crucial to reveal the hierarchicalheterogeneous structure of glioma stem/initiating cells in GBMs anddelineate the cell-cell interaction network.

Such interactions are largely mediated by soluble mediators secretedfrom different cell types. Glioma cells secrete cytokines and chemokinesto recruit and subvert their untransformed neighbor microglia that inturn produces inflammatory factors to promote tumorigenesis (Leung etal., 1997, Acta Neuropathol, 93: 518-527; Prat et al., 2000, NeurosciLett, 283: 177-180; Platten et al., 2003, Annals of Neurology, 54:388-392; Galasso et al, 2000, Experimental Neurology, 161: 85-95),reflecting the mutual paracrine stimulation between microglial cells andglioma cells. Such a complex cell-cell dialogue in a highlyheterogeneous tumor microenvironment is a paramount governing mechanismin cancer immunobiology, but remains difficult to study due to the lackof technologies that can perform informative analysis of single cellprotein profiles, in particular, the proteins secreted to modulatecell-cell communications. In certain embodiments, the present methodallows for the single cell analysis of secreted proteins from cellswithin or in the vicinity of a tumor in order to evaluate the cell-cellsignaling within the microenvironment. This can allow for thecharacterization of the tumor, including the aggressiveness, or stage ofa tumor, based on the observed signaling phenotype.

The device and method of the invention may be used to determine thepresence and/or quantity of any compound. Suitable types of compoundsinclude proteins, nucleic acids, protein fragments, surface receptors,hormones, growth factors, and the like. The precise combination ofcompounds of interest being assayed by way of the invention is easilycontrollable and defined by the eventual user. For example, detection ofa particular compound is only limited by the availability of a captureagent (e.g. antibody, peptide, nucleic acid sequence, etc.) thatspecifically binds to the compound. In certain embodiments, thecombination of compounds of interest provides the user information aboutthe phenotype of a cell contained within the microwell of the device. Inone embodiment, the device is configured for the multiplexed detectionof secretable proteins. For example, in one embodiment, the device isconfigured for the multiplexed detection of one or more of MIF, IL-1RA,IL-15, IL-13, IL-12, IL-10, IL-8, IL-7, IL-6, IL-5, IL-4, IL-3, IL-1b,IL-1a, VEGF, PDGF, NGFβ, HGF, EGF, MCSF, SCF, MIP-1b, IL-22, TNFβ, TNFα,RANTES, MCP-1, IL-17A, TSLP, IL-27, IL-27-1, MMP9, MMP2, IL-23, IL-9,GMCSF, IFN, GCSF, TGFβ, TGFα, MIP-1a, and IL-2.

As detailed herein, the device and method of the invention allows for hemultiplexed simultaneous detection of a large number of compounds. Byutilizing the spatial location/shape of isolated features and differentdetectable labels within each feature, the invention providessimultaneous detection of, in certain embodiments, up to 20, up to 30,up to 40, up to 50, up to 75, up to 100, up to 200, or more compounds.For example, FIG. 26 depicts a correlation map of 45 compounds asdetected using a device and method described herein.

Given the heterogeneity of single cells, even within a given tissue, thepresent method provides a powerful tool to quickly and effectivelydetermine the presence of a plurality of phenotypes within a population.For example, the method and device of the invention allows for thedetermination of whether all cells share the same or similar proteomic,genomic, and/or secretomic profile, or rather if there is the presenceof isolated single cells within the population which has an alteredprofile.

The method allows for the determination of distinct cellular phenotypesthat can be used, among other things, to determine the presence of aparticular harmful phenotype. Determination of profiles on a single cellbasis allows for detection of particular phenotypes whose individualprofile would be hidden in a population profile. Further, determinationof profiles based upon the multiplexed detection of a large number ofcompounds can identify phenotypes that would be hidden in methods thatonly detect one or a few compounds.

This may be used, for example, in determining the presence of cell witha profile indicative of a cancer cell. In one embodiment, the method isused to evaluate the progression of a particular disease based upon theobserved phenotypical stage of one or more single cells. In anotherembodiment, the method could be used to detect a particular type ofcancer cell or to characterize the aggressiveness or invasiveness of acancer cell. In another embodiment, the method could be used to detect acell with a phenotype indicative of a metastasizing cell. For example,the multiplexed single cell profiling described herein can be used toinvestigate the secretomic profile of single cells from a tumor, whichcan identify the presence a subset of individual cells whose profile isindicative of metastasis. This therefore would allow for very earlydiagnoses, at stages when it would otherwise be near impossible todetect.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1: High-Throughput Secretomic Analysis of Single Cells to AssessFunctional Cellular Heterogeneity

Secreted proteins dictate a range of cellular functions in human healthand disease. Because of the high degree of cellular heterogeneity and,more importantly, polyfunctionality of individual cells, there is anunmet need to simultaneously measure an array of proteins from singlecells and to rapidly assay a large number of single cells (more than1000) in parallel. Described herein is a simple bioanalytical assayplatform consisting of a large array of subnanoliter microchambersintegrated with high-density capture agent microarrays for highlymultiplexed protein detection from over a thousand single cells inparallel. This platform has been tested for both cell lines and complexbiological samples such as primary cells from patients. Distinctheterogeneity among the single cell secretomic signatures was observed,which, for the first time, can be directly correlated to the cells'physical behavior such as migration. Compared to the state-of-the-artprotein secretion assay such as ELISpot and emergingmicrotechnology-enabled assays, the approach described herein offersboth high throughput and high multiplicity. It also has a number ofclinician-friendly features such as ease of operation, low sampleconsumption, and standardized data analysis, representing a potentiallytransformative tool for informative monitoring of cellular function andimmunity in patients. Further description of the data presented hereinmay be found in Lu et al., 2013, Anal Chem, 85(4): 2518-2556, which isherein incorporated by reference in its entirety.

The materials and methods employed in the experiments are now described.

Fabrication of Capture Agent Arrays

The mold for PDMS replica is a silicon master etched with deepreactive-ion etching (DRIE) method. It was pre-treated withchlorotrimethylsilane (Aldrich) vapor overnight to facilitate PDMSrelease. PDMS prepolymer and curing agent (RTV615, Momentive) was mixedcompletely (parts A and B in 10:1 ratio) and poured onto the siliconmaster. Air bubbles were removed via vacuum desiccator for 1 h, and thePDMS was cured in the oven at 80° C. for 2 hrs. After curing, the PDMSlayer was peeled off the mold and holes for inlet and outlet ports werepunched. The device was cleaned via sonication in ethanol and 2-propanolbefore bonding with a poly-L-lysine microarray slide (Erie Scientific).The assembly was then baked in the oven at 80° C. for 2 hrs tostrengthen the bonding. The PDMS microchip for antibody flow patterningcontains 20 separate microchannels which can pattern up to 20 differentantibodies respectively. The typical width and pitch of set of lines is25 μm, 50 μm respectively in the PDMS flow patterning microchip.

For the flow patterning of the capture agent array, 1.5 μL of differentantibodies were injected into microchannels separately and flowedthrough the microfluidic channels until dry. All the antibodies used inexperiments are summarized in Table 1. Antibodies are immobilized on thepoly-L-lysine glass slide to form the capture agent array. After flowpatterning, the glass slide can be stored in the refrigerator at 4° C.,and the PDMS layer will be released before use.

TABLE 1 List of all antibodies used Primary antibody (vendor:clone)(catalog No.) Secondary antibody (vendor: clone) (catalog No.)Mouse Anti-Human IFN gamma (ebio: NIB42)(14-7318) Anti-Human IFN gammaBiotin(ebio: 4S.B3)(13-7319) Anti-Human TNF alpha Purified(ebio:MAb1)(14-7348) Anti-Human TNF alpha Biotin(ebio: MAb11) (13-7349) RatAnti-Human IL-2(ebio: MQ1-17H12)(14-7029) Rabbit Anti-Human IL-2Biotin(ebio: Polyclonal) (13-7028) Mouse Anti-Human IL-4(ebio: 8D4-8)(14-7049) Mouse Anti-Human IL-4 biotin(ebio: MP4-25D2)(13-7048)Anti-human IL-1b(ebio: CRM56)(16-7018) Mouse Anti-Human IL-1 betaBiotin(ebio: CRM57)(13-7016) Mouse Anti-Human TNF beta(ebio:359-238-8)(14-7329) Mouse Anti-Human TNF beta Biotin(ebio: 359-81-11)(13-7327) Mouse anti-human RANTES(R&D) (DY278) Goat anti-humanRANTES(R&D) (DY278) Rat Anti-Human IL-6(ebio: MQ2-13A5) (14-7068) RatAnti-Human IL-6 biotin(ebio: MQ2-39C3)(13-7068) Rat Anti-HumanIL-10(ebio: JES3-9D7)(14-7108) Rat Anti-Human IL-10 biotin(ebio:JES3-12G8)(13-7109) Mouse Anti-Human IL-12(ebio: B-T21 (BT21))(14-7128)Mouse Anti-Human IL-12 biotin(ebio: C8.6)(13-7129) Anti-HumanGMCSF(BD)(555126) Anti-Human GMCSF biotin(BD)(555126) Mouse Anti-CCL2(MCP-1)(ebio: 5D3-F7)(14-7099) Armenian Hamster Anti-CCL2 (MCP-1)Biotin(ebio: 2H5)(13-7096) Mouse Anti human EGF(R&D) (DY236) Goat Antihuman EGF Biotin (R&D) (DY236) Mouse Anti human FGF basic(R&D) (DY233)Mouse Anti human FGF basic Biotin (R&D) (DY233) Mouse Anti human HGF(R&D) (DY294) Goat Anti human HGF Biotin (R&D) (DY294) Mouse Anti humanPDGF-AB (R&D) (DY222) Goat Anti human PDGF-AB Biotin (R&D) (DY222) GoatAnti human TGF-a Biotin (R&D) (DY239) Goat Anti human TGF-a Biotin (R&D)(DY239) Mouse Anti human VEGF (R&D) (DY293B) Goat Anti human VEGF Biotin(R&D) (DY293B) Mouse Anti human MIF (R&D) (DY289) Goat Anti human MIFBiotin (R&D) (DY289) Rat Anti-Human IL-5(ebio: TRFK5)(14-7052) RatAnti-Human IL-5 biotin(ebio: JES1-5A10)(13-7059) Mouse Anti-HumanIL-13(ebio: PVM13-1)(14-7139) Rabbit Anti-Human IL-13 biotin(ebio:Polyclonal)(13-7138)

Fabrication of Sub-Nanoliter Microchamber Array Chips

The mold for the sub-nanoliter microchamber array is a silicon masteretched with DRIE method. It was also pre-treated withchlorotrimethylsilane (Aldrich) vapor overnight to facilitate PDMSrelease. The sub-nanoliter microfluidic chamber array chips for singlecell capture were fabricated out of PDMS (RTV615, Momentive, parts A andB in 10:1 ratio) using soft lithography techniques. Air bubbles wereremoved via vacuum desiccator for 1 h, and the PDMS was cured in theoven at 80° C. for 2 hrs. The sub-nanoliter microchamber array chipscontain 5044 cell capture chambers in 14 columns with approximately 550microwells per column. Image “markers” are included on the lithographymask for automated microwell and cell recognition at distinct spatiallocations on the resultant microwell array

Cell Culture and Stimulation

Human A549 cell line was cultured in F12/K medium supplemented with 10%fetal bovine serum (FBS, ATCC). Human U937 cell line was purchased fromATCC (American Type Culture Collection, ATCC) and cultured in RPMI 1640medium (Gibco, Invitrogen) supplemented with 10% fetal bovine serum(FBS, ATCC). Every 100 uL of U937 cell suspension was differentiatedwith 1 μL 20 μg/mL phorbol 12-myristate 13-acetate (Fisher) andchallenged by 1 μL 1 mg/mL lipopolysacchride (Calbiochem) to activateToll-like receptor 4 (TLR4) signaling before its suspension was pipettedonto PDMS microwell array.

Human Tissue Specimens

Human samples were obtained from individuals with meningioma. Patientprimary tissue was first minced with a scalpel and then placed in 1×TrypLE Select (Invitrogen). Tissue was triturated with fire-polishedglass pipettes and allowed to incubate for 5 mins at 37° C. The cellsuspension was triturated again with fire-polished glass pipettes andallowed to incubate for another 5 mins at 37° C. The cell suspension wasthen strained through a 40 μm cell strainer (BD Falcon) and washed withDMEM-F12 (Gibco, Invitrogen). The suspension was then centrifuged at 300RCF for 5 min. The pellet was resuspended in DMEM-F12. Red blood celllysis solution (Miltenyi Biotec) was then used to remove erythrocytesand centrifuged at 300 RCF for 5 min. The cells were then resuspended inDMEM-F12 at 10⁶ cells/mL.

Single Cell Trapping with PDMS Microwell Array

Before performing the single cell trapping experiment, the PDMSmicrowell array and capture agent array glass slide was blocked with 3%BSA solution (Sigma) respectively for 2 hrs and then rinsed with freshcell medium. Cells were suspended in fresh medium just before cellcapture. The PDMS microwell array was placed facing upward and cellculture medium solution was removed until a thin layer was remained onthe PDMS microwell array surface. Cell suspension was pipetted (50-200μL) onto the microwell array and allowed to settle for 10 mins so thatcells would fall into the microwells. The antibody glass slide was puton the top of PDMS microwell array with capture agent array resting onthe cell capture chambers. Then the PDMS microwell array and glass slidewere clamped tightly with screws and pressure was distributed bysprings. Single cells will be trapped in the microwell array and theassembly was allowed to incubate for 24 hours to allow for cellsecretion. After the trapped cells were incubated for 24 hours, thescrews were released to remove the capture agent array glass slide, andELISA immunoassay procedures were performed and the results weredetected and analyzed with Genepix scanner and software.

Population Micro Array

Cell population assay was performed on custom printed antibodymicroarray, which was spotted with a Spotbot 3 microarrayer (Arrayit) onpoly-L-lysine glass slides. Twelve identical subgroups which had thesame antibody pattern were printed on each glass slide. After printing,the antibody glass side was kept in a wet box (containing saturated NaClsolution at 75% relative humidity) for 5 hours. Before cell populationassay, the glass side was bonded with a PDMS microwell slab and blockedwith 3% BSA solution for 2 hours. Then cell culture supernatant wasadded into different microwells and allowed to incubate for 1 hour.Following incubation, ELISA immunoassay procedures are performed, andthe results were detected and analyzed with Genepix scanner andsoftware.

Immunoassay Procedures

ELISA procedures were followed to translate secreted cytokines by singlecells into detectable signals. A mixture of biotinylated detectionantibodies (Table 1) were pipetted onto the glass slide and incubatedfor 45 min at room temperature to complete the sandwich immunoassayfollowed by washing with 3% BSA solution. APC dye-labeled streptavidin(eBioscience, 200 μL, 5 μg/mL) was added onto glass slide and incubatedfor another 45 min. Following, the glass slide was washed with 3% BSAagain and blocked with 3% BSA for 0.5 hr. Following the BSA blocking,the glass slide was dipped in DPBS, DPBS, DI water, DI watersequentially and finally blown dry.

Fluorescence Detection and Analysis

Genepix 4000B and 4200A scanners (Molecular Devices) were used to obtainscanned fluorescent images for FITC and APC channels. Two color channels488 (blue) and 635 (red) were used to collect fluorescence signals. Theimage was analyzed with GenePix Pro software (Molecular Devices) byloading and aligning the microwells array template followed byextraction of fluorescence intensity values. Fluorescence results wereextracted with the image analysis tool in GenePix Pro. The fluorescenceresults were then matched to each of the chambers of the sub-nanolitermicrochamber array chips analyzed via optical imaging.

Automated Optical Image Analysis and Cell Counting

The assembly was imaged on an automatic microscope stage (Prior) toacquire optical images recording the number and location of cells ineach microwell. Automated whole chip optical images were taken with aPaxcam (Paxit!) attached to a Nikon Diaphot SA optical microscope(Nikon), and stitched together using Paxit software (Paxit!). Afterimaging, the assembly was put into an incubator at 37° C. for 24 hrs toallow for cell secretion. After 24 hours of incubation, cells are imagedagain to observe cell function. A home developed software using OpenCV(Intel) was written to automate cell counting, and the cell counts werematched with the extracted fluorescent data to their respective cellchambers.

Data and Statistical Analyses

All fluorescent scanned arrays were processed with Genepix software toextract background subtracted average fluorescent signal for allfeatures in each set. A home developed Matlab (MathWorks) code wascreated for automated extraction of fluorescent data and generation ofscatterplots. Excel (Microsoft) and OriginPro 8 (OriginLab) was used tocompile extracted data. Heatmaps and unsupervised clustering weregenerated from the extracted data using the software Cluster/Treeview(Eisen Laboratory). Statistical analysis was conducted in Excel,OriginPro 8, and R (R Development Core Team).

The results of the experiments are now described.

Described herein is a high-throughput single-cell secretomic analysisplatform that integrates a subnanoliter microchamber array andhigh-density capture agent array for simultaneous detection of 14cytokines from more than a thousand single cells in parallel. The chipcan be executed in a simple assay “kit” with no need for sophisticatedfluid handling or bulky equipment. The utility of this device isdemonstrated for analyzing the secretion of human cell lines and primarycell samples dissociated from the fresh tumor of patients. The resultsreveal that there is distinct heterogeneity among the single cellsecretomic signatures of a population and that the correlations obtainedbetween the various proteins studied are in agreement with theirfunctional classifications. This technology diverges from prior works ofantibody barcode-based protein secretion measurement technique (Ma etal., 2011, Nat Med 17(6):738-43) by using simplified schemes of cellcapture (Balaban, et al, 2004, Science 305(5690):1622-5),quantification, automated data analysis, and eliminating bulky fluidhandling systems, resulting in a truly practical and informative toolthat may find immediate use in both laboratory research and clinicaldiagnosis.

Design, Fabrication, and Assembly of a Single-Cell Secretomic AnalysisChip

The single-cell secretomic analysis device consists of two separateparts (FIG. 1A): a high-density capture agent array glass substrate forsurface-bound immunoassay and a subnanoliter microchamber array forcapture of single cells. The capture agent array slide comprises 30repeats of features, each of which contains up to 20 stripes ofdifferent antibodies, immobilized on a poly-L-lysine-coated surface. Theantibody stripes are 20 μm in width and the pitch size of a full arrayis 1 mm. The microchamber array is a one-layer microchip fabricated bysoft lithography (Unger et al., 2000, Science 288(5463):113-6) frompolydimethylsiloxane (PDMS) (Unger et al., 2000, Science288(5463):113-6), an optically transparent silicone elastomer widelyused for biological microfluidics. It contains 5440 rectangularmicrochambers, each of which is 1.8 mm, 20 μm, and 15 μm, in length,width, and depth, respectively. These two parts were manufacturedindependently and combined during the assay such that the capture agentarray slide acts as a disposable test strip and the microchamber arrayas a reusable device. To use this platform, a drop of single cellsuspension (˜10⁶ cells/mL) is directly pipetted onto the surface of themicrochamber array chip. The cells fall into the microchambers bygravity, and then the aforementioned capture agent array slide is placedantibody-side down on top of the microchambers such that the lines areperpendicular to the length of the microchambers. The microchamber isdesigned to be sufficiently long as to contain at least a full set offeatures, thereby eliminating the need for precise alignment. Finallythis assembly is fixed by two transparent plastic plates with fourspring-adjusted screws (FIG. 6) and placed in a conventional tissueincubator for single-cell secretion measurement. Proteins secreted fromindividual cells are captured by the capture agent array and read out byincubating with biotinylated detection antibodies and then streptavidinconjugated with a fluorescence probe (e.g., Cy5). As compared to theprototype single cell proteomic chip (Ma et al., 2011, Nat Med17(6):738-43), this setup does not require a sophisticated microfluidiccontrol system or any bulky equipment to operate and thus is moreamenable to widespread use by researchers and clinicians with minimalengineering background.

The high-density capture agent array is fabricated using awell-established microchannel guided patterning technique (Fan et al.,2008, Nat Biotechnol 26(12):1373-8), and in principal can be created byseveral other high-density microarray printing techniques such as inkjetprinting or nanoscale tip-based spotting. The flowpatterning chip is aseparate PDMS slab that has inlets leading to 20 individual serpentinemicrochannels in which individual antibody solutions (1 μL each) areprecisely metered, added, and flowed through all microchannels inparallel to ensure uniform loading of antibodies on the surface.Fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA)solution was used to evaluate the patterning quality. The result showssuccessful fabrication of high-density protein array across a large area(1 in.×2 in.) and excellent uniformity (<5% in fluorescent intensity) asrevealed by the fluorescence intensity line profile (FIG. 1B and FIG.7). This ensures the observed protein signal variations (>10%) from thefollowing single cell secretomic assays are attributed to cellularheterogeneity, rather than the nonuniformity of the starting captureagent array.

A motorized phase-contrast imaging system has been developed to imageall cells in the cell capture chip within 10 min (FIG. 1C), and an imageanalysis algorithm allows for identification of individual cells andtheir x/y coordinates and counting of cells in each microchamber. Thesimple microchamber array chip format was chosen because it is easy tooperate, but as a consequence it is not possible to ensure that one cellis captured per chamber. However, optimization of cell density in thestock solution readily gives rise to more than 1000 single cell chambersin a microchip (FIG. 8), permitting high-throughput analysis of singlecells.

Protein Panel and Validation

The proteins assayed by the capture agent array are listed in FIG. 2A.Assessment of these particular proteins secreted from single cells is ofparticular importance due to their functions in a range of cellularprocesses (Raman et al., 2007, Cancer Lett 256(2):137-65; Wu et al.,2012, PLoS Comput Biol 8:e1002355; Zou, 2005, Nat Rev Cancer5(4):263-74; Dranoff 2004, Nat Rev Cancer 4(1):11-22). They includecytokines, chemokines, and growth factors involved in a wide range ofimmunological or pathophysiological processes. Assessment of theseproteins secreted from single cells is of importance in the study ofcellular immunity and cell-cell signaling networks. In order tosimultaneously measure these proteins from single cells, captureantibodies are immobilized on the substrate as a high-density captureagent array. Prior to performing single-cell analysis, the assay wasvalidated using recombinant proteins. Individual recombinant protein wasspiked into fresh cell culture medium over a 4-log range ofconcentrations and exposed to the full panel of antibodies in order toassess cross-reactivity, the limit of detection (LOD), and dynamicrange. The antibodies with cross-reactivity over 5% (at 5 ng/mL ofprotein concentration) is eliminated or replaced. Ultimately a panel ofantibody pairs were obtained as summarized in Table 1. The titrationcurves (FIG. 2B) demonstrate the feasibility of quantitativemeasurements of these proteins in the multiplexed array, with a typicalmeasurement range of 3 orders of magnitude. The LOD ranged from 400pg/mL to below 10 pg/mL depending on the affinity of antibody pairs. Onthe basis of the volume of a microchamber (˜0.54 nL) and therepresentative detection sensitivity (˜10 pg/mL), the amount of proteinthat can be detected by the capture agent array in a microchamber is onthe order of 5.4 ag, which is approximately equal to ˜160 molecules.Thus, the platform has the sensitivity to detect proteins secreted froma single cell (typical copy number ˜10²⁻⁵).

Single-Cell Protein Secretomic Analysis on Cell Lines

The single-cell secretomic analysis chip was first used to measure 14proteins from a human glioblastoma multiforme cell line (U87). In thisexperiment, up to 10 cells were captured in each microchamber, with 1278of the microchambers capturing single cells. During the flow patterningof capture agent arrays, FITC-BSA (0.5 mg/mL) was always flowed inchannel 1 to form a continuous line of fluorescence signal serving asboth a position reference and an internal quality/uniformity control. Asshown in a representative region of the scanned fluorescence image (FIG.3A and FIG. 9A), both the blue FITC-BSA reference line and the redpatterned signals corresponding to protein secretion levels are readilyvisible. Shown in the same figure are a bright field image of 14microchambers with cells loaded, the corresponding fluorescent arrayimage, and an overlay of the two. The major proteins observed after 24 hof incubation (FGF, VEGF, MIF, IL-6, IL-8, and MCP-1) are mainlypro-inflammatory cytokines or chemoattractant proteins.

Automated quantitation of the fluorescence intensity of each protein ina microchamber was conducted using the image analysis software Genepix6.0. The secretomic profile for only those microchambers containingsingle cells were extracted and a heat map of the resulting secretionprofiles (FIG. 3B and FIG. 10) indicates the existence of cell-cellvariation. While the majority of cells produce IL-6 and IL-8, the levelof these proteins varies among individual cells and the secretion ofother lower abundance proteins such as MCP-1 and FGF apparently exhibitheterogeneous signatures; only a small fraction of cells express theseproteins at high levels. To verify the single cell measurements, akinetic bulk population secretion measurement was performed in parallelon the supernatant collected from the same cells, incubated over thesame time, and measured using a conventional pin-spotted microarray. Theresult (FIG. 3D and FIG. 11) also reveals FGF, VEGF, IL-6, IL-8, andMCP-1 as the top five proteins that are all consistent with single-cellanalysis although the relative levels are different. However, MIF didnot show up in the population assay. Interestingly, it was observed thatthe protein level as measured by fluorescence intensity is not alwaysproportional to the number of cells and sometimes cannot be interpretedby an additive effect (FIG. 3C and FIG. 12). A secretomic analysis chipwas loaded with many more cells, and MIF signal decreases withincreasing number of cells in the capture chambers, revealing thepossibility of paracrine signaling and the regulation of MIF with anincreasing number of cells (FIG. 12). This small level of discrepancy isexpected as the two assays are not biologically identical (for example,the bulk assay detects the end point protein profile while the singlecell assay measures accumulated signals over the period of incubation;the population arrays are subjected to paracrine signaling while singlecell measurements are not). Overall, these comparative studies are ingood agreement with each other and demonstrated the validity of thesingle cell secretomic analysis microchip. Another advantage of thisplatform is that it also measures proteins secreted from multiple cellsat the same time. While IL-6 and IL-8 secretion increases withincreasing number of cells in a microchamber, the amount of MCP-1 or MIFincrease does not change significantly when cell number exceeds 2,suggesting the existence of a possible mechanism similar to “quorumsensing” in which the paracrine mechanism in the multicellular systemcontrols homeostasis.

The single cell secretomic analysis chip was also used to measure twoadditional cell lines in order to assess the broad applicability of thisplatform. The first is an immune cell line (U937). The cells are humanmonocytes, which can be stimulated with phorbol myristate acetate (PMA)to differentiate into functional macrophage cells and then challenged bycytotoxin lipopolysaccharide (LPS) to stimulate cytokine production.This process emulates the inflammatory immune response of humanmacrophages to Gram-negative bacteria (Aderem and Ulevitch, 2000, Nature406(6797):782-7). The major proteins observed are RANTES, TNF-α, MCP-1,IL-6, and IL-8 (FIG. 9B). While the majority of cells produce RANTES,IL-8, and TNF-α, the level of these proteins varies among individualcells and the secretion of other lower abundance proteins such as MCP-1and IL-6 exhibit heterogeneous signatures. A bulk population secretionmeasurement was performed in parallel on the supernatant collected fromthe same cells to verify the single cell experiments (FIG. 13). Theresult also reveals RANTES, IL-8, MCP-1, IL-6, and TNF-α as the top fiveproteins, consistent with the single cell analysis. IL-8 and RANTESsecretion increases with an increasing number of cells in amicrochamber, and the amount of MCP-1 or TNF-α increases does not changesignificantly when cell number exceeds 4 (FIG. 9C). The second is a lungcarcinoma cell line (A549) that constitutively produces cytokines orgrowth factors. Therefore, the basal level secretion from these cellswas measured with no stimulation. The major proteins observed are MCP-1,IL-6, IL-8, VEGF, and FGF (FIG. 14) that were also validated by the bulkpopulation assay using standard pin-spotted antibody microarray assays(FIG. 15). The proteins secreted from A549 cells include bothpro-inflammatory cytokines and growth factors, in agreement with therole of lung tumor cells in both maintaining tumor growth and promotingan inflammatory microenvironment. Altogether, the cell line studiesdemonstrated that the platform is capable of rapid, quantitative, andhigh throughput analysis of protein secretion profiles in single cellscompared to current conventional methods such as ELISpot. Validating theplatform with cell lines allow the expansion of the sample repertoire toinclude more complex samples such as tissue specimens from patients.

Correlation Between Secretomic Signature and Migratory Property

Although flow cytometry-based single cell analysis allows formultiplexed protein measure, the measured protein profile cannot bedirectly correlated to the cell's behavior and activity such asmigratory property. The platform described herein utilizes live cellimaging to count captured cells, thus permitting simultaneousmeasurement of cellular behavior and subsequent correlation to thecorresponding protein profile of the same cell. Herein the migration oflung cancer cells (A549) loaded in the single cell secretomic analysischip was measured by measuring the distance of movement beforeincubation and after 24 h of incubation (FIG. 4A). These cells were seento adhere to the channel wall and migrate at varying speeds. The resultsare summarized in a heatmap showing single-cell secretomic profilessorted by increasing cell migration distance (FIG. 16). P-value analysisof cytokine levels was performed in high motility (top 20% over theobserved range) vs low motility (bottom 20% of the same range) cells.While the majority of the cells do not migrate, the highly migratorycells are statistically associated with high expression of IL-8 (P<0.01)(FIG. 4B). The correlation between the secretion of MCP-1 and cellmigration was less significant (FIG. 4c,d ). IL-6 appears to benegatively associated with cell motility in the scatter plots but doesnot show statistical correlation using the aforementioned test. Theseproteins have been linked to the increase of motility and metastaticpotential in different cancers (Singh et al., 1994, Cancer Res54(12):3242-7; Li et al., 2003, J Immunol 170(6):3369-76; Waugh andWilson, 2008, Clin Cancer Res 14(21):6735-41), and through theinvestigation of single cell IL-8 secretion, it may be possible to studythe secretomic signatures of individual cells linked to metastasis. Inbrief, the platform for the first time shows simultaneous measurement ofprotein secretomic signature and phenotypic properties (e.g., migration)of single live cells that can lead to improved understanding of cellularfunctions and the underlying molecular mechanisms.

Secretomic Profiling of Single Tumor Cells from Clinical PatientSpecimens

To expand the utility of the platform to measuring multiplexed secretionin cells derived from complex biospecimens, our device was also appliedto the measurement of fresh primary tumor tissue from three patients(Table 2) with a malignant brain tumor, glioblastoma multiforme(patients 1 and 2), or meningioma (patient 3). A portion (<0.2 g) of thesurgically resected tumor tissue is washed with ice coldphosphate-buffered saline, minced into smaller fractions and thendissociated into a single cell suspension using collagenase (FIG. 5A).The cells were spun down and resuspended in medium at a density of ˜10⁶cells/mL. Within 1 h of tissue procurement, the single cell suspensionis loaded onto the single-cell secretomic analysis device via pipette.After allowing the cells to secrete cytokines for 12 h, the pattern onthe array is developed with detection antibodies and scanned. A rawfluorescent image (FIG. 5B, patient 1) shows excellent protein signalsand similar background compared to the scanned image from cell lines.The antibody array includes 14 proteins as shown in FIG. 5b . In thisexperiment, between 0 and 22 cells were captured within a microchamber,with 1058 of the microchambers capturing single cells. The fluorescenceintensities of each secreted cytokine from each individual channel wasquantified and a heat map of the single cell secretion profiles was thengenerated (FIG. 5C). Unsupervised hierarchical clustering of the singlecell secretion profiles resolved three separate populations of cellswith varying activity. One cluster of cells (FIG. 5C, blue cluster) wasgenerally more active, secreting a wider range of proteins presumablycorresponding to a more aggressive phenotype, while the cells indicatedby green exhibit the lowest level of cytokine production and mayrepresent more quiescent phenotypes such as tumor stem/progenitor cells(Wicha et al., 2006, Cancer Res 66(4):1883-90). The large fractionindicated by orange are a variety of functional phenotypes. The resultfrom the patient 2 (FIG. 5D) shows similarities to the results frompatient 1, such as MIF and IL-8 as major proteins but a differentpattern in that it has a much reduced production of inflammatorycytokines and a higher level of EGF. The second tier proteins all showdistinct cellular heterogeneity. FIG. 17 and FIG. 18 present histogramsand scatter plots of individual proteins, which show both the relativelevels of proteins and the distributions among the cell population.

TABLE 2 Summary of the patient medical records Sample Patient codeGender Age Tumor type Grade Location 1 RFa Female 64 Glioblastoma 4 Left10-28-11 frontal 2 RFa Female 66 Glioblastoma 4 Right 06-11-12 side notspecified 3 RFa Female 47 Transitional 1 Right 04-02-12 Meningioma side

Pseudo-three-dimensional scatter plots of the single cell cytokinemeasurements for the patient primary tumors in the format of flowcytometric plots were compiled and a 14×14 mosaic matrix was formed(FIG. 5E). The proteins are shown at the diagonal line, and each panelis a pairwise correlation plot, for each of which we performed a linearregression analysis to yield the R value. Then the whole matrix iscolor-coded by red (positive correlation) and blue (negativecorrelation), and the color intensity is proportional to R. In thepatient 1 matrix, all the inflammatory cytokines are apparentlyassociated within one cluster and several growth factors are grouped ina separate cluster, reflecting their functional difference. In theresult for patient 2, the pro-inflammatory cytokines, although generallyexpressed at low levels, also show intercorrelation. Interesting, thesecretion of EGF is negatively correlated to proinflammatory andchemoattactant proteins (MCP-1, GMCSF, and IL-8). A third sample from apatient (patient 3) with transitional meningioma was also analyzed,which is considered a more homogeneous and less inflammatory tumor.Indeed the results for patient 3 (FIG. 19 and FIG. 20) show reducedpro-inflammatory cytokine signals. These studies imply the relevance ofthe results to these cells' physiological functions or pathologicalcondition. Currently surgical treatment remains the most effectivetherapy of human glioblastoma. Afterward, chemotherapy might be carriedout systemically or by putting drug-containing wafers into the surgicalcavity to further eradicate invasive tumor cells that have diffused tonormal brain tissue (Lesniak and Brem, 2004 Nat Rev Drug Discovery3(6):499-508). The platform potentially can distinguish and quantitateinvasive cell phenotypes as they generally produce more cytokines aswell as different profile of cytokines, which has the clinical value todetermine tumor invasiveness and tailor the chemotherapeutic strategyfor individual patients. In addition, these proteins that act as thesoluble signals to mediate cell-cell communication in tumormicroenvironment may be identified as new therapeutic targets forpersonalized treatment (Dvorak 2002, J Clin Oncol 20(21):4368-80; Richand Bigner, 2004, Nat Rev Drug Discovery 3(5):430-46; Reardon and Wen,2006, Oncologist 11(2):152-64).

Single Cell Proteomics

Single cell proteomic analysis has generally been much more challengingthan genetic analysis from single cells, due to the lack of equivalentamplification methods for proteins such as polymerase chain reaction(PCR) for nucleic acids. Recent advance in flow cytometric analysisallows for 34-plexed measurement of protein markers from single cells,but most proteins are either surface receptors or cytoplasmic proteins(Bendall et al., 2011, Science 332(6030):687-96). Intracellular cytokinestaining (ICS) enables indirect assessment of “secreted” proteins, butcurrently the number of cytokines that can be measured is practicallylimited to below 10, presumably due to increased nonspecific bindingfrom a large number of antibodies in the limited volume of a singlecell. Further, increasing the number of compounds detected using ICS iscorrelated with decreased accuracy and increased noise due to spectraloverlap between individual labels. Moreover, unlike protein secretion,it is not a direct measurement of cell function. Thus, multiplexedprotein secretion measurement is a missing piece of functionalcharacterization of single cells. It has become increasingly evidentthat even genetically homogeneous cells can be extremely heterogeneous,leading to many unanswered questions in studying their biology (Bendallet al., 2011, Science 332(6030):687-96). Studying the secretion profileof single cells can reveal much more about tumor heterogeneity thanstudying the signaling patterns of cells in a population wherein thesignals become averaged out and all defining information is lost,emphasizing the need for studying single cell secretion (Bendall andNolan, 2012, Nat Biotechnol 30(7):639-47; Michor and Polyak, 2010,Cancer Prev Res 3(11):1361-4).

Described herein is a sub-nanoliter multiplexed immunoassay chip thatenables high throughput, simultaneous detection of a panel of 14cytokines secreted from over a thousand single cells in parallel. Thisplatform provides significant advantages specific to the detection ofsecreted proteins and offers information complementary to that obtainedthrough flow cytometry. An example scenario where this device wouldoffer unique advantages is that when a cell separation tool is used tosort out a phenotypically identical cell population using specificsurface markers, these cells can then be placed in the present device tofurther reveal cellular heterogeneity at the functional level. Forinstance, human T cell lineages often display a number of functions, andthe complex combinations of multiple functions in a single T cellsdictates the “quality” of this cell in response to a specific antigen.Recent studies showed that multifunctional T cells often exhibit greaterpotency and durability (Seder et al., 2008, Nat Rev Immunol8(4):247-58). The latest HIV vaccine trials employed the ELISpottechnique to count interferon-γ (IFN-γ)-secreting T cells as a means toassess the efficacy of vaccination, but it turns out that mostIFN-γ-secreting cells are terminally differentiated effector T cells andhave minimal protective effect against viral infection. This platformrepresents a promising tool to perform polyfunctional analysis on thecells isolated from a flow cytometer or other separation techniques,e.g., magnetically assisted cell sorting (Adams et al., 2008, Proc NatlAcad Sci USA 105(47):18165-70), to bring single-cell protein assay toanother level of functional analysis. A potential concern of theplatform is that cells are isolated in the sealed microchamber and mayexperience a condition that affects the normal functioning of primarycells ex vivo. As a bioanalytical tool, the microchip was not intendedto perform long-term culture of cells and the typical assay time is afew hours to 1 day. It has been reported that ex vivo assay of primarycells in a sealed and isolated environment do produce proteins over along time as anticipated for their intrinsic physiological activity (Maet al., 2011, Nat Med 17(6):738-43); Han et al., 2012, Proc Natl AcadSci USA 109(5):1607-12) and interestingly the cells could gain greaterviability in a sealed nanoliter-chamber because it recapitulates the invivo crowdedness in primary tissue and retains sufficient concentrationsof cytokines for more effective autocrine signaling. Thus, the microchipis a promising platform for high-throughput analysis of proteinsecretion profiles from single primary cells and may assist indifferential diagnosis and monitoring of cellular functions in patients.

Example 2: Spatial and Spectral Encoded 45-Plex Assay

Described herein is the development of one embodiment of the device ofthe invention which uses the spatial position and color of detectablelabels to assay for the presence of up to 45 different compounds ofinterest. The use of differently labeled secondary antibodies allows forthe ability to vastly increase the number of compounds of interest thatcan be simultaneously detected.

FIG. 27 is a diagram depicting how the assay functions. Each spatialline (or other isolated feature) has three different primary antibodies,each specific for the detection of three different compounds ofinterest. The detection antibodies (secondary antibodies) used toprovide a detectable label at the site of compound binding, are eachlabeled with a differently colored fluorescent label. Analysis of theassay thus comprises using a multi-color laser scanning method toobserve the spatial location of each detectable label throughout thedevice.

FIGS. 28 and 29 demonstrate the ability to detect 3 different colorsfrom the device. FIG. 28 shows an overlay of the microwells with theantibody array. On the right, the same field of view is imaged for thethree different wavelengths. The detection at a specific location of aspecific wavelength thus determines the presence of a particularcompound of interest. FIG. 29 demonstrates that a number of differentwavelengths can be used and shows the uniformity of wavelengthdetection. Further, as shown in FIG. 29, the device provides uniformprotein coding across the slide.

An exemplary panel is shown in FIG. 30, which demonstrates that 14different lines are used, each with 3 differently colored labels todetect 42 compounds of interest. Also included is a fifteenth line usedas a control. The antibodies used in this assay are given below in Table3.

TABLE 3 Antibodies used in 45-plex spatial and spectral assay Captureantibody Detection antibody Protein Isotope/clone/vendor/catalogIsotope/clone/vendor/catalog IL-1a Mouse IgG2A/4414/RD/MAB200 MouseIgG1, κ/364-3B3-14/Biolegend/500104 IL-1b MouseIgG1/JK1B-1/Biolegend/508202 Mouse IgG2b, κ/JK1B-2/Biolegend/508304 IL-3Mouse IgG1/653A10B1/Invitrogen/AHC0832 Rat IgG1,κ/BVD8-3G11/Biolegend/500502 IL-4 Mouse IgG1, κ/8D4-8/Biolegend/500702Rat IgG1, κ/MP4-25D2/Biolegend/500802 IL-5 Rat IgG2a,κ/JES1-39D10/Biolegend/500902 Rat IgG2a, κ/JES1-5A10/Biolegend/501006IL-6 Rat IgG2a, κ/MQ2-39C3/Biolegend/501204 Rat IgG1,κ/MQ2-13A5/Biolegend/501102 IL-7 Rat IgG2a,κ/BVD10-11C10/Biolegend/506604 Rat IgG1, κ/BVD10-40F6/Biolegend/501302IL-8 Mouse IgG1, κ/H8A5/Biolegend/511502 Mouse IgG1,κ/E8N1/Biolegend/511402 IL-10 Rat IgG2a, κ/JES3-12G8/Biolegend/501504Rat IgG1, κ/JES3-9D7/Biolegend/501402 IL-12(p70) MouseIgG1/24945/RD/MAB611 Rat IgG1, κ/7B12/Biolegend/511002 IL-13 MouseIgG1/32116/RD/MAB213 Rat IgG1, κ/JESI0-5A2/Biolegend/501902 IL-15 MouseIgG/RD duoset capture antibody/DY247 MouseIgG1/AM00959PU-N/Acris/AM00959PU-N IL-1RA MouseIgG1/JK1RA-1/Biolegend/509902 Mouse IgG/RD duoset Capture antibody/DY280MIF Mouse IgG1 Kappa/2A10-4D3/Abnova/H00004282-M01 Mouse IgG1Kappa/2A10-4D3/Abnova/H00004282-M01 IL-17A Mouse IgG2a,κ/BL127/Biolegend/512603 Mouse IgG1, κ/BL23/Biolegend/512702 MCP-1 MouseIgG1, κ/5D3-F7/Biolegend/502607 Armenian HamsterIgG/2H5/Biolegend/505902 Rantes Mouse IgG2b kappa/VL1/Invitrogen/AHC1052Mouse IgG1/21418/RD/MAB678 TNF-a Mouse IgG1, κ/Mab11/Biolegend/502902Mouse IgG1, κ/MAb1/Biolegend/502802 TNF-b Mouse IgG2A/5807/RD/MAB621Mouse IgG1, κ/359-238-8/Biolegend/503002 IL-22 Rat IgG2a,kappaIL22JOP/Ebioscience/16-7222-85 Mouse IgG1/142928/RD/MAB7821 MIP-1bMouse IgG1 kappa/A174E18A7/Invitrogen/AHC6114 MouseIgG2B/24006/RD/MAB271 SCF Mouse IgG1/J231/Peprotech/500-M44 MouseIgG1/13302/RD/MAB655 M-CSF Mouse IgG2b/AM09180PU-N/Acris/AM09180PU-NMouse IgG2A/21113/RD/MAB616 EGF Mouse IgG1/AM09146PU-N/Acris/AM09146PU-NMouse IgG1/10827/RD/MAB636 HGF Mouse IgG1/SBF5 C1.7/Novus/NB100-2696Mouse IgG1/24516/RD/MAB694 NGF-b Mouse IgG1, κ/JKhNGF-1/biolegend/509602Mouse IgG1, κ/JKmNGF-1/Biolegend/509702 PDGF RabbitIgG/polyclonal/Acris/PP1061P2 Mouse IgG1/108128/RD/MAB1739 VEGF MouseIgG1/A183C/Invitrogen/AHG0114 Mouse IgG2B/26503/RD/MAB293 IL-2 MouseIgG2A/5355/RD/MAB602 Goat IgG/RD duoset detection antibody/DY202 MIP-1aMouse IgG1/14D7 1G7/Invitrogen/AHC6034 Goat IgG/RD duoset detectionantibody/DY270 TGF-a Goat IgG/RD duoset capture antibody/DY239 GoatIgG/RD duoset detection antibody/DY239 TGF-b BD 559119 BD 559119 G-CSFMouse IgG1/3316/RD/MAB214 Goat IgG/polyclonal/RD/BAF214 IFN-g MouseIgG1, κ/MD-1/Biolegend/507502 Mouse IgG1,kappa/4S.B3/ebioscience/13-7319-85 GMCSF Rat IgG2a,κ/BVD2-23B6/Biolegend/502202 Rat IgG2a, κ/BVD2-21C11/Biolegend/502304IL-9 Ebioscience Ready sets go Ebioscience Ready sets go IL-23Ebioscience Ready sets go Ebioscience Ready sets go MMP-2 Mouse IgG/RDduoset capture antibody/DY902 Mouse IgG/RD duoset detectionantibody/DY902 MMP-9 Mouse IgG1/36020/RD/MAB936 GoatIgG/polyclonal/RD/BAF911 IL-27 Ebioscience Ready sets go EbioscienceReady sets go IL-29 Ebioscience Ready sets go Ebioscience Ready sets goTSLP Ebioscience Ready sets go Ebioscience Ready sets go BSA -488 488conjugated BSA conjugated BSA-532 532 conjugated BSA conjugated BSA-635635 conjugated BSA conjugated

Experiments were run to ensure that the antibodies on the same spatialline are all below threshold in cross-reactivity. In these experiments,the antibody array was subjected to a solution containing only EGF. Asshown in FIG. 31, using the spatial and spectral detection schemedescribed, EGF is the only species detected. It was observed that thelocation and color corresponding to EGF lights up significantly abovethe 1000 intensity threshold that is used as background. This ensuresthat any observed responses are only caused by the particular cytokinebinding event of interest. This data proves that, based on spectraldata, that the triple spatial encoding in the spectral assay is viableand will not produce significant error in terms of quantification of thesecretomic cytokines.

For calibration, titration curves (FIG. 32A-C) were determined for eachof the compounds of interest, in order to properly quantitativelycorrelate a detected intensity signal to the concentration of thecompound of interest in the sample.

Experiments were conducted using human macrophage cells derived fromU937 cell lines through the differentiation by 50 ng/mL PMA for 48hours. Experiments compared cells that were starved with FBS free mediumto cells that were fed with 10% FBS. Starved and non-starved cells wereeither left non-stimulated or alternatively, were stimulated with 10μg/mL LPS, a common pathogenic molecule in gram-negative bacteria.Responses detected by the spatial/spectral assay described herein werecompared to micro-ELISA. Detection of compound binding was conducted asdescribed elsewhere herein, but was repeated for the 3 wavelengths used.FIG. 33 depicts the raw data for the 488 nm wavelength, FIG. 34 depictsthe raw data for the 532 wavelength, and FIG. 35 depicts the raw datafor the 635 nm wavelength. For ease of analysis, the detection of the 3wavelengths were “extended” as shown in FIG. 36, which shows thedetection of all the species of interest (i.e. combined species detectedin the 488 nm, 532 nm and 635 nm raw data). As shown, the data wasartificially pseudo-colored to a single color (in this case red), forease of subsequent analysis. The single cell analysis using thespatial/spectral assay was compared to population-based micro-ELISAassay, the data of which is shown in FIG. 37.

Comparing the average single-cell data from the spatial/spectral assayto the population-based micro-ELISA data revealed important differencesin these analyses (FIG. 38). In certain instances, there was a greaterthan 10× difference in the predicted ‘phenotype’ or ‘secretome’. Forexample, IL12 and IL10 are predicted to be much higher (˜10×) in theirsecretions when looking at the whole population data compared to theaveraged single cell data. This indicates that micro-ELISA does notaccurately evaluate the single-cell response. These large differencesmay be due to sub-populations with distinct polyfunctionality thatcannot be shown on the population level, or population based sensingdifferences (i.e. cell to cell communication), or different cytokinesecretion patterns based on time stamp of analysis.

The difference between starved and non-starved is validated against themicro-ELISA technique within high agreement for most cytokines. Inparticular IL-8, MIP-1b, MCP-1, IL-1RA, GMCSF, GCSF, MIP-1a, IL-1b,IFN-g: correlate well (statistically significant); MIF, MMP-9, TSLP,Rantes: Show significant population decrease in averaged single cell;IL-6, IL-10, TNF-a: doesn't correlate well due to perhaps differences inevaluation discussed above.

FIG. 39 depicts the results of the experiments of using the 45-plexedsingle cell assay comparing single-cell secretions of non-stimulatedversus LPS (100 ng/mL) stimulated cells. Data is presented as ahistogram (top), heat maps (middle) and 2-d bar graph of the averagedsignals (bottom).

The single cell multiplexing array assay (SCMA) was also validatedagainst intracellular cytokine staining (ICS) (FIG. 40 and FIG. 41). Itwas seen that the “secretions” as detected by ICS was increased comparedto the single cell secretion assay described herein. Without wishing tobe bound by any particular theory, these increases in ICS may be due tothe fact that this detection are predicted rather than actually secretedproteins.

FIG. 42 depicts a type of data output that describes thepolyfunctionality of the cells (non-stimulated versus LPS stimulated).As depicted, the graphs demonstrate the number of cytokines detected persingle cell.

Example 3: IsoPlexis: A 45-Plex Single-Cell Secretion Profiling Platformto Interrogate Functional Cellular Heterogeneity

ELISpot including its variant FLUOROSpot is the only technology widelyused to measure true secretion of immune effector proteins from singlecells and thus remains the mainstay of pre-clinical and clinical tool toassess cellular immunity. However it can measure only one or twoproteins and provides minimal biological information. Therefore it failsto reveal the immune cell quality and protective ability because it isnot able to capture the complete picture of functional diversity oridentify the most potent polyfunctional population. A technology thatcan measure an array of proteins from single cells is highly desired andwill help address a host of important biological and medical questionsranging from immune diversity, intratumor heterogeneity,multifunctionality, to cell-cell communication network. Such technologyneeds to meet all the following requirements. (i) Single cellsensitivity: proteins can be isolated and sensitively measured from asingle cell. (ii) High multiplicity: a large panel (35 or more) ofproteins can be simultaneously measured. (iii) High content: thousandsof single cells can be analyzed in parallel. Described herein is a newtechnology that combines spatial multiplexing and spectral multiplexingin a sub-nanoliter chamber-based single-cell protein secretion assay. Itallows for simultaneous quantification of 45 secreted proteins incontrast to only 1-2 proteins using existing ELISpot assays, which isthe highest recorded multiplexing capability in single-cell proteomicassay. This platform was applied to the study of human macrophage cellsin response to pathogenic ligands, LPS, poly-IC and PAM3, that activatethree toll-like receptors (TLRs), respectively. Single-cell, high-plexprotein profiles reveal unexpectedly large cell-to-cell variability anda three-tiered response, which was further confirmed by a compressedclustering analysis using viSNE. viSNE is a recently developed analysisbased on the t-Distributed Stochastic Neighbor Embedding (t-SNE)algorithm and is a powerful tool to enable visualization, cluster ofhigh dimensional single cell data and uncover phenotypic heterogeneitybetween cells (Amir et al, 2013, Nature Biotechnology, 31: 545-552,which is incorporated herein by reference in its entirety). Threefunctional cell subsets were identified, and the response is stronglycorrelated to the initial state. The first population diminished orreturned to the indolent state. The second population remains largelyunchanged in terms of their effector functions. The third population isstrongly correlated to the elevated production of cytokines and appearsto be highly multifunctional. This three-tiered response prevails inboth macrophage cell lines and primary monocyte-derived macrophages, andappears to be an intrinsic non-genetic heterogeneity that determines thequality (multifunctionality) and extent (fraction) of cellular immuneresponse to pathogenic ligands. The study presented herein demonstratesthe ability of single-cell, high-plex protein profiling to reveal deepfunctional phenotype and heterogeneous responses to perturbagens thatcannot be probed by existing technologies. This platform has greatpotential in both preclinical and clinical studies to evaluate cellularheterogeneity, for example, in the immune system or tumormicroenvironment.

The materials and methods employed in these experiments are nowdescribed.

Fabrication of Antibody Features

The mold for PDMS replica is a silicon master etched with deepreactive-ion etching (DRIE) method. It was pre-treated withchlorotrimethylsilane (Aldrich) vapor overnight to facilitate PDMSrelease. PDMS prepolymer and curing agent (RTV615, Momentive) was mixedcompletely (parts A and B in 10:1 ratio) and poured onto the siliconmaster. Air bubbles were removed via vacuum desiccator for 1 h, and thePDMS was cured in the oven at 80° C. for 2 hrs. After curing, the PDMSlayer was peeled off the mold and holes for inlet and outlet ports werepunched. The device was cleaned via sonication in ethanol and 2-propanolbefore bonding with a poly-L-lysine microarray slide (Erie Scientific).The assembly was then baked in the oven at 80° C. for 2 hrs tostrengthen the bonding. The PDMS microchip for antibody flow patterningcontains 20 separate microchannels which can pattern up to 20 differentsolutions respectively. The typical width and pitch of the set offeatures is 25 μm, 50 μm respectively in the PDMS flow patterningmicrochip.

For the flow patterning of the antibody features, 2 μL of differentantibody mixtures (Table 3 and FIG. 30) were injected into microchannelsseparately and flowed through the microfluidic channels until dry. Allthe antibodies used in experiments are summarized in Table 3. Antibodiesare immobilized on the poly-L-lysine glass slide to form the antibodypatterned feature. After flow patterning, PDMS layer is released and theglass slide is blocked with 3% BSA(Sigma) and be stored in therefrigerator at 4° C. until use.

Fabrication of Microchamber Array Chips

The mold for the microchamber array is a silicon master etched with DRIEmethod. It was also pre-treated with chlorotrimethylsilane (Aldrich)vapor overnight to facilitate PDMS release. The microfluidic chamberarray chips for single cell capture were fabricated out of PDMS (RTV615,Momentive, parts A and B in 10:1 ratio) using soft lithographytechniques. Air bubbles were removed via vacuum desiccator for 1 h, andthe PDMS was cured in the oven at 80° C. for 2 hrs.

Antibody Conjugation

The 488 nm group and 532 nm group detection antibodies were covalentlyconjugated with Alexa fluor 488 and Alexa fluor 532 dyes respectivelyfollowing the protocol provided by the supplier. The 635 nm groupdetection antibodies are all tagged with biotin, which can be read outusing a fluorophore APC or Cy5 conjugated streptavidin or avidin throughthe binding of streptavidin/avidin with biotin.

Cell Culture and Stimulation

Human U937 cell line was purchased from ATCC (American Type CultureCollection, ATCC) and cultured in RPMI 1640 medium (Gibco, Invitrogen)supplemented with 10% fetal bovine serum (FBS, ATCC). The U937 cellswere differentiated with 50 ng/mL phorbol 12-myristate 13-acetate(Fisher) for 48 hrs. Media was then changed and replaced with normalmedium for 36 hrs. The cells were harvested with trypsin for single cellexperiment. The cells were challenged with 100 ng/mL lipopolysacchride(Calbiochem) to activate Toll-like receptor 4 (TLR4) (or otheractivation reagents like PAM3, poly IC) signaling just before itssuspension was pipetted onto PDMS microwell array.

Intracellular Cytokine Staining

Cells are harvested and seeded into tissue culture petri dish in 10⁶density with both control and treated cells. After 2 hrs, the secretioninhibitor Brefeldin A (Biolegend) was added. The cells are thenincubated for 22 hrs before harvested for intracellular flow cytomery.Cells were fixed and stained according to manufacturers' instructions.

Single Cell Trapping with PDMS Microwell Array

Before performing the single cell trapping experiment, the PDMSmicrowell array and antibody-containing glass slide were blocked with 3%BSA solution (Sigma) respectively for 2 hrs and then rinsed with freshcell medium. Cells were suspended in fresh medium just before cellcapture. The PDMS microwell array was placed facing upward and cellculture medium solution was removed until a thin layer was remained onthe PDMS microwell array surface. Cell suspension was pipetted (50-200μL) onto the microwell array and allowed to settle for 10 mins so thatcells would fall into the microwells. The antibody glass slide was puton the top of PDMS microwell array with antibodies resting facing thecell capture chambers. Then the PDMS microwell array and glass slidewere clamped tightly with screws. Single cells are thus trapped in themicrowell array and the assembly was allowed to incubate to allow cellsto secrete proteins. After the trapped cells were incubated for 24hours, the screws were released to remove the antibody glass slide, andELISA immunoassay procedures were performed and the results weredetected and analyzed with Genepix scanner and software.

Population Micro Array

Cell population assay was performed on custom printed antibodymicroarray, which was spotted with a Spotbot 3 microarrayer (Arrayit) onpoly-L-lysine glass slides. Twelve identical subgroups which had thesame antibody pattern were printed on each glass slide. After printing,the antibody glass side was kept in a wet box (containing saturated NaClsolution at 75% relative humidity) for 5 hours. Before cell populationassay, the glass side was bonded with a PDMS microwell slab and blockedwith 3% BSA solution for 2 hours. Then cell culture supernatant wasadded into different microwells and allowed to incubate for 1 hour.Following incubation, ELISA immunoassay procedures are performed, andthe results were detected and analyzed with Genepix scanner andsoftware.

Immunoassay Procedures

ELISA procedures were followed to translate cytokines secreted by singlecells into detectable signals. A mixture of biotinylated detectionantibodies (Table 3) were pipetted onto the glass slide and incubatedfor 1 hr at room temperature to complete the sandwich immunoassayfollowed by washing with 3% BSA solution. APC dye-labeled streptavidin(eBioscience, 200 μL, 5 μm/mL) was added onto glass slide and incubatedfor another 30 min. Following, the glass slide was washed with 3% BSAagain and blocked with 3% BSA for 0.5 hr. Following the BSA blocking,the glass slide was dipped in DPBS, DPBS, DI water, DI watersequentially and finally blown dry.

Fluorescence Detection and Analysis

Genepix 4200A scanners (Molecular Devices) were used to obtain scannedfluorescent images for FITC and APC channels. Three color channels 488(blue), 532 (green) and 635 (red) were used to collect fluorescencesignals. The image was analyzed with GenePix Pro software (MolecularDevices) by loading and aligning the microwells array template followedby extraction of fluorescence intensity values. Fluorescence resultswere extracted with the image analysis tool in GenePix Pro. Thefluorescence results were then matched to each of the chambers of thesub-nanoliter microchamber array chips analyzed via optical imaging.

Automated Optical Image Analysis and Cell Counting

The assembly was imaged on Nikon Eclipse Ti microscope with an automaticmicroscope stage to acquire optical images (both darkfield and obliqueview) recording the number and location of cells in each microwell. Thedarkfield image will be used to define the location and sequence of eachmicrochamber and oblique image will be used to define the cell numbersand their locations. Both images can be processed in Nikon software(NIS-Elements Ar Microscope Imaging Software) by defining threshold oneach image to realize automated cell counting. The cell counts will thenbe matched with the extracted fluorescent data to their respective cellchambers.

Data and Statistical Analyses

All fluorescent scanned slides were processed with Genepix software toextract average fluorescent signal for all features in each set. A homedeveloped Matlab (MathWorks) code was created for automated extractionof fluorescent data and generation of scatterplots. Excel (Microsoft)and OriginPro 8 (OriginLab) was used to compile extracted data. Heatmapsand unsupervised clustering were generated from the extracted data usingthe software Cluster/Treeview (Eisen Laboratory). Statistical analysiswas conducted in R (R Development Core Team).

The results of the experiments are now described.

Experiments were conducted using the 45-plexed single-cell proteinsecretion profiling platform, described herein (FIG. 43). A cellsuspension is pipetted onto the surface of the PDMS microwell arraychip, during which the cells fall into the microchambers by gravity.Then the flow patterned 45-plexed antibody immobilized glass slide isplaced facing down on top of the microchambers to ensure antibodyfeatures are perpendicular to microchambers. Zero to dozens of cells aretrapped in microchambers during this process. The captured cell numberfits into Poisson distribution. Finally this assembly is clampedtogether by two transparent plastic plates with six screws. A motorizedphase-contrast microscope is used to image the assembly to record thecell number and location in each microchamber. It is then placed in atissue culture incubator for 24 hours for cells to secret proteins.Proteins secreted from individual cells during this period are capturedby the antibodies, transformed into detectable signals by reacting withcorresponding detection antibodies afterwards and read out byfluorescence scanner finally. The fluorescence signals are quantified,corresponded with each single cell and analyzed to be presented byheatmap, scatterplot or VISNE.

The cross-talk or spectral overlap between the 488 and 532 channels wasaccounted for. Due to spectral overlap between Alexa fluor 488 and 532dyes, this glass slide will get fluorescence signal from both 488channel (real signal) and 532 channel (crosstalk). The 488 channelsignal and 532 channel signal showed good and stable correlation betweeneach other (R2≈98%) and compensation equation can be extracted (FIG.47A). Similar results were observed when observing a 532 channel realsignal and 488 channel cross talk (FIG. 47B).

The differentiation of U937 monocytes with PMA was evaluated bydetecting expression of macrophage markers CD11b (FL4) and Cd14 (FL2)(FIG. 48).

Experiments were conducted to examine U937 monocyte derived macrophagepopulation cells protein secretion results from different substratesincluding 96 well plate, PDMS in 5:1, 10:1, 20:1 ratio respectively. Theresults shows similar fold of change in different substrates for bothhigh level secretion proteins like IL-8, MCP-1, IL-6 and low levelsecretion proteins like IL-1a, IL-3, IL-4 (FIG. 49).

The heat map of secretion can be visualized as organized by the amountof cells contained with the chamber (FIG. 50). The signal from the 0cell chambers can be used as a threshold (average signal plus two timesstandard deviation) for positive secretion. The data was analyzed toexamine the correlation between secretion and cell number (FIG. 52).Experiments were also conducted to compare single cell secretion resultsbetween two different chips (FIG. 51), which demonstrates that the chipsproduce very similar results.

The device was used to analyze the secretion of proteins from U937macrophages. The dynamics of protein secretion over the 48 hoursincubation is demonstrated in FIG. 55, which shows that differentproteins have different secretion dynamics. FIG. 44 demonstratesstimulated or unstimulated (control) protein secretion from U937macrophages using various methods. For example, FIG. 44A depicts acomparison of U937 derived macrophage single cell protein secretionresults with its population cells secretion results. Generally speaking,these two results showed good correlation with each other.Interestingly, several differences were also observed. For example, IL-8intensity was observed to be very similar between control andstimulation cells in population result. However, the single cellresults, observed using the described single-cell array demonstratedthat LPS stimulated cells in fact have much higher secretion frequenciesthan control cells. This was also validated with intracellular cytokinestaining (FIG. 44B). A comparison of protein secretion frequencyobtained from single cell secretion platform and ICS (intracellularcytokine staining) is depicted in FIG. 44B.

Similar cell subpopulation definitions defined by IL-8 and MCP-1 proteinsecretion results was observed both by SCMA and ICS (FIG. 44C). Thesingle cell secretion platform was used to examine polyfunctionality ofcells. U937 macrophage single cell polyfuncationality analysis based ontheir protein secretion results demonstrated that a wide variety ofsingle cell polyfunctionality was observed and U937 derived macrophagecells showed more polyfunctionality upon the activation of TLR-4 via LPSstimulation (FIG. 44D).

Polyfunctionality of the cells is further demonstrated in FIG. 53, whichdemonstrates a wide variety of single cell poyfunctionality andincreased polyfunctionality upon LPS stimulation. This result wasverified in three independent experiments.

The results of single cell secretion analysis of macrophage responsefollowing stimulation of the TLR4 ligand, LPS is demonstrated in FIG.45. Heatmaps show the comparison between untreated and LPS stimulatedU937 monocyte derived macrophage protein secretion profiles (FIG. 45A).VISNE was used to visualize single cell secretion results (FIG. 45B).VISNE transforms high-dimensional single cell data into two dimensions,but still retains the high-dimensional structure of the original data.It visualizes individual cells similar to a scatter plot, in which allpairwise distances in high dimension are utilized to localize eachcell's position in the plot. The X and Y axis are arbitrary numbersshowing the 2D location. From this analysis it can be observed thatuntreated and LPS stimulated U937 single cells can be grouped into 3subpopulations, based upon their protein secretions (in accordance withconventional cluster analysis (FIG. 54)). This is further illustrated inFIG. 45C, where individual proteins (MIF, IL-8, MCP-1, RANTES, MIP-1a,MIP-1b) secretion results with VISNE. It is clearly demonstrated thatdifferent proteins characterize the different subgroups of cells. Forexample, some proteins, such as IL-8, MIP-1b are sensitive to LPSstimulation, while some, such as MIF, are not.

Analysis of identical cells before and after stimulation with LPSdemonstrates how each individual cell may have a specific secretionprofile. For example, FIG. 56 includes a series of plots demonstratinghow individual cells respond to LPS stimulation. This data demonstratesthat while secretion of some proteins is similar across cells, secretionof other proteins may vastly differ among the different single cells ofthe study population.

Experiments were performed to examine macrophage response to differentTLR ligands (LPS, PAM3, or poly IC) stimulation. FIG. 46A depicts theheatmaps showing protein secretion profiles of untreated and stimulated(LPS, PAMP, or poly IC) U937 monocyte derived macrophage. Further, thefrequency of cell secretion of each of the given proteins was analyzedunder each condition (FIG. 46B). Single cell results were visualizedwith VISNE, from which it was observed that both untreated andstimulated single cells are grouped into 3 subpopulations based upontheir secretions (FIG. 46C). This was further illustrated when observingthe secretion of individual proteins (MIF, IL-8, MCP-1, and MIP-1b) withVISNE (FIG. 46D).

The presently described system provides for effective determination ofcell secretion at the single cell level, which can identify importantsingle cell differences within cell populations. The benefits of thepresent system are listed in FIG. 57. There are several differences incomparing the single cell secretion assay presented here and ICS. First,it is important to note that protein expression does not equal proteinsecretion. The present single cell assay measures the amount of proteinsecreted by a cell, whereas ICS measures the protein blocked within thecell. Further, during ICS, a Golgi blocker is used which may alter cellfunction, and is toxic to cells. Conversely the present single cellassay uses a physical barrier to block signals, which is non-toxic.Additionally, the present single cell assay uses a sandwich immunoassayformat which provides greater specificity than ICS, since two differentepitopes of the protein must be recognized in order to provide a signalin sandwich technique. Thus, detection threshold for a positive cell canbe very different in these two systems.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variation.

1. A device for the multiplexed detection of a plurality of compoundsfrom single cells comprising: a microwell array comprising a pluralityof individual microwells in uniform arrangement, at least some of theplurality of individual microwells having a length of greater than 50 μmand configured to contain an isolated single cell in a sub-nanolitervolume of contents; and a capture agent array comprising a plurality ofimmobilized capture agents, each immobilized capture agent capable ofspecifically binding to one of the plurality of compounds, where theimmobilized capture agents are arranged in uniform capture agent sets,wherein each capture agent set comprises a plurality of isolatedfeatures at spatially identifiable locations, each isolated featurecomprising at least one immobilized capture agent; wherein the microwellarray and capture agent array are coupled to form a plurality ofenclosed interfaces, each enclosed interface comprising a microwell anda capture agent set such that the contents of each microwell areaccessible to all of the isolated features of at least one set, therebyaccessible to all of the immobilized capture agents.
 2. The device ofclaim 1, wherein each of the plurality of isolated features has adistinguishable spatial localization. 3-4. (canceled)
 5. The device ofclaim 1, wherein at least some of the plurality of microwells is a highaspect ratio rectangular well, having dimensions of about 1-2 mm inlength and about 5-50 μm in depth.
 6. (canceled)
 7. The device of claim1, wherein the plurality of compounds comprise at least one proteinsecreted from a single cell contained within a microwell.
 8. The deviceof claim 1, wherein the plurality of capture agents comprise at leastone compound selected from the group consisting of an antibody, protein,peptide, peptide fragment, and nucleic acid. 9-10. (canceled)
 11. Thedevice of claim 1, wherein each capture agent set comprises about 10-100isolated features, each isolated feature comprising at least oneimmobilized capture agent that specifically binds to one compound.12-13. (canceled)
 14. The device of claim 1, wherein the capture agentarray comprises greater than 10 different capture agents, therebyallowing for the detection of greater than 10 different compounds.15-16. (canceled)
 17. A method of spatially encoded multiplexeddetection of a plurality of compounds from a single cell, the methodcomprising: providing a microwell array comprising a plurality ofindividual microwells in uniform arrangement; applying a fluid to asurface of the microwell array such that a sub-nanoliter volume of thefluid comprising a single cell flows into at least one microwell;providing a capture agent array comprising a plurality of immobilizedcapture agents, each capture agent capable of specifically binding toone of the plurality of compounds, where the immobilized capture agentsare arranged in capture agent sets, wherein each capture agent setcomprises a plurality of isolated features at spatially identifiablelocations, each isolated feature comprising at least one immobilizedcapture agent; contacting the microwell array with the capture agentarray to form a plurality of enclosed interfaces, each enclosedinterface comprising a microwell and a capture agent set such that thefluid within each microwell is accessible to all of the isolatedfeatures of a set and is thereby accessible to all of the of immobilizedcapture agents; providing suitable conditions to allow for the bindingof the plurality of compounds to the immobilized capture agents to formimmobilized capture agent-compound complexes; contacting the captureagent array with a plurality of labeled secondary capture agents,wherein each labeled secondary capture agent specifically binds to aformed immobilized capture agent-compound complex, to form immobilizedcapture agent-compound-labeled secondary capture agent complexes;detecting the presence of the detectable label on the capture agentarray; and correlating the presence of the detectable label on thecapture agent array with the presence of at least one compound. 18-22.(canceled)
 23. The method of claim 17, wherein the plurality ofcompounds comprise at least one protein secreted from a single cellcontained within a microwell.
 24. The method of claim 17, wherein theplurality of capture agents comprise at least one compound selected fromthe group consisting of an antibody, protein, peptide, peptide fragment,and nucleic acid.
 25. The method of claim 17, wherein at least oneisolated feature comprises more than one immobilized capture agent,wherein each immobilized capture agent within the isolated feature hasan associated secondary capture agent with a different detectable label.26. The method of claim 17, wherein each microwell is rectangular with alength of about 10-2000 μm, a width of about 10-100 μm, and a depth ofabout 10-100 μm. 27-29. (canceled)
 30. The method of claim 17, whereinthe capture agent array comprises greater than 10 different captureagents, thereby allowing for the detection of greater than 10 differentcompounds. 31-32. (canceled)
 33. The method of claim 17, whereinapplying the fluid to the microwell array surface produces a pluralityof individual microwells which comprise a single cell. 34-38. (canceled)39. The method of claim 17, wherein the method assays the phenotype of aplurality of single cells within the sample by detecting 5 or morecompounds secreted by the single cells. 40-72. (canceled)
 73. A systemfor the multiplexed detection of a plurality of compounds from singlecells comprising: a device comprising: a microwell array comprising aplurality of individual microwells in uniform arrangement, at least someof the plurality of individual microwells configured to contain a singlecell in a sub-nanoliter volume of contents; and a capture agent arraycomprising a plurality of immobilized capture agents, each immobilizedcapture agent capable of specifically binding to one of the plurality ofcompounds, where the immobilized capture agents are arranged in uniformcapture agent sets, wherein each capture agent set comprises a pluralityof isolated features at spatially identifiable locations, each isolatedfeature comprising at least one immobilized capture agent; wherein themicrowell array and capture agent array are coupled to form a pluralityof enclosed interfaces, each enclosed interface comprising a microwelland a capture agent set such that the contents of each microwell areaccessible to all of the isolated features of at least one set, therebyaccessible to all of the immobilized capture agents; and a plurality ofsecondary capture agents, wherein each secondary capture agentscomprises a detectable label and is configured to bind to a immobilizedcapture agent-compound complex formed at an isolated feature by thebinding of a compound of the plurality of compounds to an immobilizedcapture agent of the plurality of immobilized capture agents. 74-91.(canceled)